Tomato hybrid ‘vespolino’

ABSTRACT

The invention relates to a new and distinctive tomato hybrid, designated ‘Vespolino,’ to the plants of tomato hybrid ‘Vespolino,’ to the plant parts of tomato hybrid ‘Vespolino’ including the fruit, and for producing a hybrid tomato plant by crossing tomato hybrid ‘Vespolino’ with itself or another tomato line. The invention further relates to methods for producing a tomato plant containing in its genetic material one or more transgenes and to the transgenic plants produced by that method and to the methods for producing other tomato lines derived from tomato hybrid ‘Vespolino.’

BACKGROUND OF THE INVENTION

The present invention relates to a new and distinctive tomato hybriddesignated ‘Vespolino.’ All publications cited in this application areherein incorporated by reference.

There are numerous steps in the development of any novel, desirableplant germplasm. Plant breeding begins with the analysis and definitionof problems and weaknesses of the current germplasm, the establishmentof program goals, and the definition of specific breeding objectives.The next step is selection of germplasm that possess the traits to meetthe program goals. The goal is to combine in a single variety or hybridan improved combination of desirable traits from the parental germplasm.These important traits may include higher yield, field performance,fruit and agronomic quality such as firmness, color, content in solublesolids, acidity and viscosity, resistance to diseases and insects, andtolerance to drought and heat. With mechanical harvesting of the tomatofruits for process purpose, i.e., juice, paste, catsup, etc., uniformityof plant characteristics such as germination, growth rate, maturity, andplant uniformity is also important.

Practically speaking, all cultivated and commercial forms of tomatobelong to a species most frequently referred to as Lycopersiconesculentum Miller. Lycopersicon is a relatively small genus within theextremely large and diverse family Solanaceae which is considered toconsist of around 90 genera, including pepper, tobacco, and eggplant.The genus Lycopersicon has been divided into two subgenera, theesculentum complex which contains those species that can easily becrossed with the commercial tomato and the peruvianum complex whichcontains those species which are crossed with considerable difficulty(Stevens, M., and Rick, C. M., Genetics and Breeding, In The TomatoCrop, A scientific basis for improvement, pp. 35-109, Atherton, J.,Rudich, G. (eds.), Chapman and Hall, New York (1986)). Due to its valueas a crop, L. esculentum Miller has become widely disseminated all overthe world. Even if the precise origin of the cultivated tomato is stillsomewhat unclear, it seems to come from the Americas, being native toEcuador, Peru, and the Galapagos Islands, and initially cultivated byAztecs and Incas as early as 700 AD. Mexico appears to have been thesite of domestication and the source of the earliest introduction. It isthought that the cherry tomato, L. esculentum var. cerasiforme, is thedirect ancestor of modern cultivated forms.

Tomato is grown for its fruit, widely used as a fresh market orprocessed product. As a crop, tomato is grown commercially whereverenvironmental conditions permit the production of an economically viableyield. In California, the first largest process market and secondlargest fresh market in the United States, processing tomatoes areharvested by machine. The majority of fresh market tomatoes areharvested by hand at vine ripe and mature green stages of ripeness.Fresh market tomatoes are available in the United States year round.Process tomato season in California is from late June to September.Process tomatoes are used in many forms, as canned tomatoes, tomatojuice, tomato sauce, puree, paste, and catsup. Over the 500,000 acres oftomatoes that are grown annually in the United States, approximately 40%are grown for fresh market consumption, the balance are grown forprocessing.

Tomato is a simple diploid species with twelve pairs of differentiatedchromosomes. The cultivated tomato is self-fertile and almostexclusively self-pollinating. The tomato flowers are hermaphrodites.Commercial cultivars were initially open-pollinated. Most have now beenreplaced by better yielding hybrids. Due to its wide dissemination andhigh value, tomato has been intensively bred. This explains why such awide array of tomatoes are now available. The size may range from smallto large, and there are cherry, plum, pear, standard, and beefsteaktypes. Tomatoes may be grouped by the amount of time it takes for theplants to mature fruit for harvest. In general, the cultivars areconsidered to be early, midseason or late-maturing. Tomatoes can also begrouped by the plant's growth habit; determinate or indeterminate.Determinate plants tend to grow their foliage first, then set flowersthat mature into fruit if pollination is successful. All of the fruittend to ripen on a plant at about the same time. Indeterminate tomatoesstart out by growing some foliage, then continue to produce foliage andflowers throughout the growing season. These plants will tend to havetomato fruit in different stages of maturity at any given time. Morerecent developments in tomato breeding have led to a wider array offruit color. In addition to the standard red ripe color, tomatoes can becreamy white, lime green, pink, yellow, golden, or orange.

Choice of breeding or selection methods depends on the mode of plantreproduction, the heritability of the trait(s) being improved, and thetype of cultivar used commercially (e.g., F₁ hybrid cultivar, purelinecultivar, etc.). For highly heritable traits, a choice of superiorindividual plants evaluated at a single location will be effective,whereas for traits with low heritability, selection should be based onmean values obtained from replicated evaluations of families of relatedplants. Popular selection methods commonly include pedigree selection,modified pedigree selection, mass selection, and recurrent selection.

The complexity of inheritance influences choice of the breeding method.Backcross breeding is used to transfer one or a few favorable genes fora highly heritable trait into a desirable cultivar. This approach hasbeen used extensively for breeding disease-resistant cultivars. Variousrecurrent selection techniques are used to improve quantitativelyinherited traits controlled by numerous genes. The use of recurrentselection in self-pollinating crops depends on the ease of pollination,the frequency of successful hybrids from each pollination, and thenumber of hybrid offspring from each successful cross.

Each breeding program should include a periodic, objective evaluation ofthe efficiency of the breeding procedure. Evaluation criteria varydepending on the goal and objectives, but should include gain fromselection per year based on comparisons to an appropriate standard,overall value of the advanced breeding lines, and number of successfulcultivars produced per unit of input (e.g., per year, per dollarexpended, etc.).

Promising advanced breeding lines are thoroughly tested and compared toappropriate standards in environments representative of the commercialtarget area(s) for three years at least. The best lines are candidatesfor new commercial cultivars; those still deficient in a few traits areused as parents to produce new populations for further selection.

These processes, which lead to the final step of marketing anddistribution, usually take from eight to twelve years from the time thefirst cross is made. Therefore, development of new cultivars is atime-consuming process that requires precise forward planning, efficientuse of resources, and a minimum of changes in direction.

A most difficult task is the identification of individuals that aregenetically superior, because for most traits the true genotypic valueis masked by other confounding plant traits or environmental factors.One method of identifying a superior plant is to observe its performancerelative to other experimental plants and to a widely grown standardcultivar. If a single observation is inconclusive, replicatedobservations provide a better estimate of its genetic worth.

The goal of tomato breeding is to develop new, unique, and superiortomato inbred lines and hybrids. The breeder initially selects andcrosses two or more parental lines, followed by repeated selfing andselection, producing many new genetic combinations. The breeder cantheoretically generate billions of different genetic combinations viacrossing, selfing, and mutations. The breeder has no direct control atthe cellular level. Therefore, two breeders will never develop the sameline, or even very similar lines, having the same tomato traits.

Each year, the plant breeder selects the germplasm to advance to thenext generation. This germplasm is grown under unique and differentgeographical, climatic, and soil conditions, and further selections arethen made, during and at the end of the growing season. The inbred lineswhich are developed are unpredictable. This unpredictability is becausethe breeder's selection occurs in unique environments, with no controlat the DNA level (using conventional breeding procedures), and withmillions of different possible genetic combinations being generated. Abreeder of ordinary skill in the art cannot predict the final resultinglines he develops, except possibly in a very gross and general fashion.The same breeder cannot produce the same line twice by using the exactsame original parents and the same selection techniques. Thisunpredictability results in the expenditure of large research monies todevelop a superior new tomato hybrid line.

The development of commercial tomato hybrids requires the development ofhomozygous inbred lines, the crossing of these lines, and the evaluationof the crosses. Pedigree, backcross, or recurrent selection breedingmethods are used to develop inbred lines from breeding populations.Breeding programs combine desirable traits from two or more hybrid linesor various broad-based sources into breeding pools from which hybridlines are developed by selfing and selection of desired phenotypes. Thenew inbreds are crossed with other inbred lines and the hybrids fromthese crosses are evaluated to determine which have commercialpotential.

Pedigree breeding is used commonly for the improvement ofself-pollinating crops or inbred lines of cross-pollinating crops. Twoparents which possess favorable, complementary traits are crossed toproduce an F₁. An F₂ population is produced by selfing one or severalF₁s or by intercrossing two F₁s (sib mating). Selection of the bestindividuals is usually begun in the F₂ population; then, beginning inthe F₃, the best individuals in the best families are selected.Replicated testing of families, or hybrid combinations involvingindividuals of these families, often follows in the F₄ generation toimprove the effectiveness of selection for traits with low heritability.At an advanced stage of inbreeding (i.e., F₆ and F₇), the best lines ormixtures of phenotypically similar lines are tested for potentialrelease as new cultivars or new parents for hybrids.

Mass and recurrent selections can be used to improve populations ofeither self- or cross-pollinating crops. A genetically variablepopulation of heterozygous individuals is either identified or createdby intercrossing several different parents. The best plants are selectedbased on individual superiority, outstanding progeny, or excellentcombining ability. The selected plants are intercrossed to produce a newpopulation in which further cycles of selection are continued.

Backcross breeding has been used to transfer genes for a simplyinherited, highly heritable trait into a desirable homozygous cultivaror inbred line which is the recurrent parent. The source of the trait tobe transferred is called the donor parent. The resulting plant isexpected to have the attributes of the recurrent parent (e.g., cultivar)and the desirable trait transferred from the donor parent. After theinitial cross, individuals possessing the phenotype of the donor parentare selected and repeatedly crossed (backcrossed) to the recurrentparent. The resulting plant is expected to have the attributes of therecurrent parent (e.g., cultivar) and the desirable trait transferredfrom the donor parent.

Descriptions of other breeding methods that are commonly used fordifferent traits and crops can be found in one of several referencebooks (e.g., “Principles of Plant Breeding” John Wiley and Son, pp.115-161 (1960); Allard (1960); Simmonds (1979); Sneep et al. (1979);Fehr (1987)).

Proper testing should detect any major faults and establish the level ofsuperiority or improvement over current cultivars. In addition toshowing superior performance, there must be a demand for a new cultivarthat is compatible with industry standards or which creates a newmarket. The introduction of a new cultivar will incur additional coststo the seed producer, the grower, processor and consumer; for specialadvertising and marketing, altered seed and commercial productionpractices, and new product utilization. The testing preceding release ofa new cultivar should take into consideration research and developmentcosts, as well as technical superiority of the final cultivar. Forseed-propagated cultivars, it must be feasible to produce seed easilyand economically.

Once the inbreds that give the best hybrid performance have beenidentified, the hybrid seed can be reproduced indefinitely as long asthe homogeneity of the inbred parent is maintained. A single-crosshybrid is produced when two inbred lines are crossed to produce the F₁progeny.

Tomato is an important and valuable field crop. Thus, a continuing goalof tomato plant breeders is to develop stable, high yielding tomatohybrids that are agronomically sound. The reasons for this goal areobviously to maximize the amount of fruit produced on the land used, aswell as to improve the fruit qualities. To accomplish this goal, thetomato breeder must select and develop tomato plants that have thetraits that result in superior parental lines for producing hybrids.

SUMMARY OF THE INVENTION

The present invention provides a novel tomato hybrid designated‘Vespolino.’ This invention thus relates to the seeds of ‘Vespolino,’ tothe plants of ‘Vespolino’ and plant parts of ‘Vespolino,’ to methods forproducing a tomato plant containing in its genetic material one or moretransgenes, and to the transgenic tomato plants produced by that method.This invention also relates to methods for producing other tomato linesderived from ‘Vespolino’ and to the tomato lines derived by the use ofthose methods. This invention further relates to hybrid tomato seeds andplants produced by crossing line ‘Vespolino’ with another tomato line.

The invention discloses methods of vegetatively propagating a plant ofthe present invention and plants produced by such methods. Thisinvention also discloses methods for producing a fruit of a tomato plantof the present invention and fruits produced by such methods.

The tomato plant of the invention may further comprise, or have, acytoplasmic factor or other factor that is capable of conferring malesterility. Male sterility may also be provided by nuclear genes such asthe recessive ms gene. Parts of the tomato plant of the presentinvention are also provided, such as, e.g., fruits and pollen obtainedfrom a hybrid plant and an ovule of the hybrid plant.

In another aspect, the present invention provides regenerable cells foruse in tissue culture of tomato hybrid ‘Vespolino.’ The tissue culturewill preferably be capable of regenerating plants having thephysiological and morphological characteristics of the foregoing hybridtomato plant, and of regenerating plants having substantially the samegenotype as the foregoing hybrid tomato plant. Preferably, theregenerable cells in such tissue cultures will be embryos, protoplasts,meristematic cells, callus, pollen, leaves, anthers, pistils, stems,petioles, roots, root tips, fruits, seeds, flowers, cotyledons,hypocotyls, or the like. Still further, the present invention providestomato plants regenerated from the tissue cultures of the invention.

Another objective of the invention is to provide methods for producingother tomato plants derived from tomato hybrid ‘Vespolino.’ Tomato linesderived by the use of those methods are also part of the invention.

The invention also relates to methods for producing a tomato plantcontaining in its genetic material one or more transgenes and to thetransgenic tomato plant produced by that method.

In another aspect, the present invention provides for single geneconverted plants of ‘Vespolino.’ The single transferred gene maypreferably be a dominant or recessive allele. Preferably, the singletransferred gene will confer such traits as male sterility, herbicideresistance, insect resistance, resistance for bacterial, fungal, orviral disease, male fertility, improved harvest characteristics,enhanced nutritional quality, modified fruit yield, or improvedprocessing characteristics. The single gene may be a naturally occurringtomato gene or a transgene introduced through genetic engineeringtechniques.

The invention further provides methods for developing a tomato plant ina tomato plant breeding program using plant breeding techniquesincluding recurrent selection, backcrossing, pedigree breeding,restriction fragment length polymorphism enhanced selection, geneticmarker enhanced selection, and transformation. Seeds, tomato plants, andparts thereof, including the fruit, produced by such breeding methodsare also part of the invention.

DEFINITIONS

In the description and tables that follow, a number of terms are used.In order to provide a clear and consistent understanding of thespecification and claims, including the scope to be given such terms,the following definitions are provided:

Allele. The allele is any of one or more alternative form of a gene, allof which alleles relates to one trait or characteristic. In a diploidcell or organism, the two alleles of a given gene occupy correspondingloci on a pair of homologous chromosomes.

Attachment point. The point on the tomato fruit where the fruit isconnected to the tomato plant.

Backcrossing. Backcrossing is a process in which a breeder repeatedlycrosses hybrid progeny back to one of the parents, for example, a firstgeneration hybrid F₁ with one of the parental genotype of the F₁ hybrid.

BRIX. Means a percentage by weight of the fruit of sugar in solutionmeasured using a refractometer, wherein the fruit is cut in half and thejuice within the fruit is squeezed onto a lens. The juice on the lens isthen measured by the refractometer.

Determinate tomato. A tomato variety that comes to fruit all at once,then stops bearing. Often referred to as a “bush” variety sincedeterminate tomato plants tend to be compact. Determinate varieties arebest suited for commercial growing since they can be harvested all atonce.

Essentially all the Physiological and morphological characteristics. Aplant having essentially all the physiological and morphologicalcharacteristics means a plant having the physiological and morphologicalcharacteristics, except for the characteristics derived from theconverted gene.

Flesh color. The color of the tomato flesh that can range fromorange-red to dark red when at ripe stage (harvest maturity).

Fruit. A ripened ovary, together with any other structures that ripenwith the ovary and form a unit.

Gene converted. Gene converted or conversion plant refers to plantswhich are developed by a plant breeding technique called backcrossingwherein essentially all of the desired morphological and physiologicalcharacteristics of an inbred are recovered in addition to the one ormore genes transferred into the inbred via the backcrossing technique orvia genetic engineering.

Indeterminate tomato. A tomato variety that grows and produces fruituntil killed by frost. An indeterminate tomato variety can have flowers,immature fruit, and mature fruit on the same plant, at the same timethroughout the growing season. Often referred to as “vining” sinceindeterminate tomato plants can grow very long and usually need cagingor staking for support.

pH. The pH is a measure of acidity. A pH under 4.35 is desirable toprevent bacterial spoilage of finished products. pH rises as fruitmatures.

Plant part. A plant part means any part of a plant including, but notlimited to, cell, protoplast, embryo, pollen, ovule, flower, leaf, stem,cotyledon, hypocotyl, meristematic cell, root, root tip, pistil, anther,shoot tip, shoot, fruit, and petiole.

Regeneration. Regeneration refers to the development of a plant fromtissue culture.

Relative maturity. Relative maturity is an indication of time until atomato genotype is ready for harvest. A genotype is ready for harvestwhen 90% or more of the tomatoes are ripe.

Semi-erect habit. A semi-erect plant has a combination of lateral andupright branching and has an intermediate-type habit between a prostateplant habit, having laterally growing branching with fruits most of thetime on the ground, and an erect plant habit having branching goingstraight up with fruit being off the ground.

Soluble Solids. Soluble solids refers to the percent of solid materialfound in the fruit tissue, the vast majority of which is sugars. Solublesolids are directly related to finished processed product yield ofpastes and sauces. Soluble solids are estimated with a refractometer,and measured as degrees brix.

Quantitative Trait Loci (QTL). Quantitative trait loci refer to geneticloci that control to some degree numerically representable traits thatare usually continuously distributed.

Uniform ripening. Refers to a tomato that ripens uniformly, i.e., onethat has no green discoloration on the shoulders. The uniform ripeningis controlled by a single recessive gene.

Vegetative propagation. Means taking part of a plant and allowing thatplant part to form roots where plant part is defined as leaf, pollen,embryo, cotyledon, hypocotyl, meristematic cell, root, root tip, pistil,anther, flower, shoot tip, shoot, stem, fruit, and petiole.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides a tomato hybrid ‘Vespolino’ with superiorcharacteristics. It produces small size fruits with an overall obovateshape. In addition, tomato hybrid ‘Vespolino’ is suitable for greenhousecultivation and the fruits are intended for fresh market or garden use.

‘Vespolino’ has shown uniformity and stability for the traits, withinthe limits of environmental influence for the traits. It has beenproduced and tested a sufficient number of generations with carefulattention to uniformity of plant type. The hybrid has been increasedwith continued observation for uniformity of the parent lines. Novariant traits have been observed or are expected in tomato hybrid‘Vespolino.’

‘Vespolino’ has the following morphologic and other characteristics(based primarily on data collected at Enkhuizen, The Netherlands).

TABLE 1 VARIETY DESCRIPTION INFORMATION Plant: Growth type:Indeterminate Plant height: Long Time of Maturity: Early Type ofculture: Under glass, staked Main use: Fresh market or garden Leaf:Division of blade: Bipinnate Intensity of green color: Medium Peduncle:Abscission layer: Present Fruit: Size: Small, about 23 g Shape inlongitudinal section: Circular Ribbing at peduncle end: Absent or veryweak Number of locules: Two or three Green shoulder (before maturity):Present Color at maturity: Red Firmness: Medium Fruit shelf-life:Medium, about 22 days Disease and pest resistance: Sensitivity tosilvering: Tolerant Meloidogyne incognita (root-knot nematode):Resistant Veritcillium dahliae race 0: Absent Fusarium oxysporum f. sp.lycopersici race 0 Present (race 1, U.S.): Fusarium oxysporum f. sp.lycopersici race 1 Present (race 2, U.S.): Fusarium oxysporum f. sp.lycopersici race 2 Absent (race 3, U.S.): Fusarium oxysporium f. sp.radicis lycopersici Absent Tomato Mosaic Virus (ToMV) strain 0: PresentTomato Mosaic Virus (ToMV) strain 1: Present Tomato Mosaic Virus (ToMV)strain 2: Present Phytophthora infestans: Absent Tomato Yellow Leaf CurlVirus (TYLCV): Absent Tomato Spotted Wilt Virus (TSWV): Absent Oidiumlycopersicum (powdery mildew): Absent

Tomato hybrid ‘Vespolino’ is similar to tomato variety ‘Caprese.’ Whilesimilar to tomato variety ‘Caprese,’ there are significant differencesas shown in Table 2. Column 1 of Table 2 shows the plant characteristic,column 2 shows the characteristic in the present invention, ‘Vespolino,’and column 3 shows the characteristic in tomato hybrid ‘Caprese.’

TABLE 2 Comparison of Characteristics Between ‘Vespolino’ and ‘Caprese’Characteristic ‘Vespolino’ ‘Caprese’ Fruit size (grams) 23 g 30 g Headstrength Stronger Normal strength Leaf length Longer Normal length

Further Embodiments of the Invention

This invention also is directed to methods for producing a tomato plantby crossing a first parent tomato plant with a second parent tomatoplant wherein either the first or second parent tomato plant is tomatohybrid ‘Vespolino.’ Further, both first and second parent tomato plantscan come from the tomato hybrid ‘Vespolino.’ Still further, thisinvention also is directed to methods for producing a‘Vespolino’-derived tomato plant by crossing ‘Vespolino’ with a secondtomato plant and growing the progeny seed, and repeating the crossingand growing steps with the ‘Vespolino’-derived plant from zero to seventimes. Thus, any such methods using ‘Vespolino’ are part of thisinvention: selfing, backcrosses, hybrid production, crosses topopulations, and the like. All plants produced using ‘Vespolino’ as aparent are within the scope of this invention, including plants derivedfrom ‘Vespolino.’ Advantageously, ‘Vespolino’ is used in crosses withother, different, tomato hybrids to produce first generation (F₁) tomatohybrid seeds and plants with superior characteristics.

It should be understood that the hybrid can, through routinemanipulation of cytoplasmic or other factors, be produced in amale-sterile form. Such embodiments are also contemplated within thescope of the present claims.

As used herein, the term plant includes plant cells, plant protoplasts,plant cell tissue cultures from which tomato plants can be regenerated,plant calli, plant clumps, and plant cells that are intact in plants orparts of plants, such as embryos, pollen, ovules, flowers, leaves,stems, and the like.

As it is well known in the art, tissue culture of tomato can be used forregeneration of tomato plants. Tissue cultures of tomato andregeneration of plants therefrom are well known and published. By way ofexample, a tissue culture comprising organs has been used to produceregenerated plants as described in Girish-Chandel et al., Advances inPlant Sciences, 13: 1, 11-17 (2000); Costa et al., Plant Cell Report,19: 3 327-332 (2000); Plastira et al., Acta Horticulturae, 447, 231-234(1997); Zagorska et al., Plant Cell Report, 17: 12 968-973 (1998);Asahura et al., Breeding Science, 45: 455-459 (1995); Chen et al.,Breeding Science, 44: 3, 257-262 (1994); Patil et al., Plant and Tissueand Organ Culture, 36: 2, 255-258 (1994). It is clear from theliterature that the state of the art is such that these methods ofobtaining plants are conventional in the sense that they are routinelyused and have a very high rate of success. Thus, another aspect of thisinvention is to provide cells which upon growth and differentiationproduce tomato plants having the physiological and morphologicalcharacteristics of tomato hybrid ‘Vespolino.’

A tomato plant can also be propagated vegetatively. A part of the plant,for example, a shoot tissue, is collected and a new plant is obtainedfrom the part. Such part typically comprises an apical meristem of theplant. The collected part is transferred to a medium allowingdevelopment of a plantlet, including, for example, rooting ordevelopment of shoots, or is grafted onto a tomato plant or a rootstockprepared to support growth of shoot tissue. This is achieved usingmethods well known in the art. Accordingly, in one embodiment, a methodof vegetatively propagating a plant of the present invention comprisescollecting a part of a plant according to the present invention, e.g., ashoot tissue, and obtaining a plantlet from said part. In oneembodiment, a method of vegetatively propagating a plant of the presentinvention comprises: a) collecting tissue of a plant of the presentinvention; and b) rooting said proliferated shoots to obtain rootedplantlets. In one embodiment, a method of vegetatively propagating aplant of the present invention comprises: a) collecting tissue of aplant of the present invention; b) cultivating said tissue to obtainproliferated shoots; and c) rooting said proliferated shoots to obtainrooted plantlets. In one embodiment, such method further comprisesgrowing a plant from said plantlets. In one embodiment, a fruit isharvested from said plant.

The advent of new molecular biological techniques has allowed theisolation and characterization of genetic elements with specificfunctions, such as encoding specific protein products. Scientists in thefield of plant biology developed a strong interest in engineering thegenome of plants to contain and express foreign genetic elements, oradditional, or modified versions of native or endogenous geneticelements in order to alter the traits of a plant in a specific manner.Any DNA sequences, whether from a different species or from the samespecies, which are inserted into the genome using transformation, arereferred to herein collectively as “transgenes.” In some embodiments ofthe invention, a transgenic variant of tomato hybrid ‘Vespolino’ maycontain at least one transgene but could contain at least 1, 2, 3, 4, 5,6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24,25, 26, 27, 28, 29, or 30 transgenes. Over the last fifteen to twentyyears several methods for producing transgenic plants have beendeveloped, and the present invention also relates to transgenic variantsof the claimed tomato hybrid ‘Vespolino.’

One embodiment of the invention is a process for producing tomato hybrid‘Vespolino’ further comprising a desired trait, said process comprisingtransforming a tomato hybrid ‘Vespolino’ plant of with a transgene thatconfers a desired trait. Another embodiment is the product produced bythis process. In one embodiment the desired trait may be one or more ofherbicide resistance, insect resistance, disease resistance, decreasedphytate, or modified fatty acid or carbohydrate metabolism. The specificgene may be any known in the art or listed herein, including, apolynucleotide conferring resistance to imidazolinone, sulfonylurea,glyphosate, glufosinate, triazine, benzonitrile, cyclohexanedione,phenoxy proprionic acid and L-phosphinothricin, a polynucleotideencoding a Bacillus thuringiensis polypeptide, a polynucleotide encodingphytase, FAD-2, FAD-3, galactinol synthase or a raffinose syntheticenzyme, or a polynucleotide conferring resistance to nematodes, brownstem rot, Phytophthora root rot, or tobacco mosaic virus.

Numerous methods for plant transformation have been developed, includingbiological and physical plant transformation protocols. See, forexample, Miki et al., “Procedures for Introducing Foreign DNA intoPlants” in Methods in Plant Molecular Biology and Biotechnology, Glick,B. R. and Thompson, J. E. Eds. (CRC Press, Inc., Boca Raton (1993) pp.67-88 and Armstrong, “The First Decade of Maize Transformation: A Reviewand Future Perspective” (Maydica 44:101-109 (1999)). In addition,expression vectors and in vitro culture methods for plant cell or tissuetransformation and regeneration of plants are available. See, forexample, Gruber et al., “Vectors for Plant Transformation” in Methods inPlant Molecular Biology and Biotechnology, Glick, B. R. and Thompson, J.E. Eds. (CRC Press, Inc., Boca Raton (1993) pp. 89-119.

A genetic trait which has been engineered into the genome of aparticular tomato plant may then be moved into the genome of anothervariety using traditional breeding techniques that are well known in theplant breeding arts. For example, a backcrossing approach is commonlyused to move a transgene from a transformed tomato variety into analready developed tomato variety, and the resulting backcross conversionplant would then comprise the transgene(s).

Various genetic elements can be introduced into the plant genome usingtransformation. These elements include, but are not limited to genes,coding sequences, inducible, constitutive, and tissue specificpromoters, enhancing sequences, and signal and targeting sequences. Forexample, see the traits, genes, and transformation methods listed inU.S. Pat. No. 6,118,055.

Plant transformation involves the construction of an expression vectorwhich will function in plant cells. Such a vector comprises DNAcomprising a gene under control of, or operatively linked to, aregulatory element (for example, a promoter). The expression vector maycontain one or more such operably linked gene/regulatory elementcombinations. The vector(s) may be in the form of a plasmid and can beused alone or in combination with other plasmids to provide transformedtomato plants using transformation methods as described below toincorporate transgenes into the genetic material of the tomato plant(s).

Expression Vectors for Tomato Transformation: Marker Genes

Expression vectors include at least one genetic marker operably linkedto a regulatory element (a promoter, for example) that allowstransformed cells containing the marker to be either recovered bynegative selection, i.e., inhibiting growth of cells that do not containthe selectable marker gene, or by positive selection, i.e., screeningfor the product encoded by the genetic marker. Many commonly usedselectable marker genes for plant transformation are well known in thetransformation arts, and include, for example, genes that code forenzymes that metabolically detoxify a selective chemical agent which maybe an antibiotic or an herbicide, or genes that encode an altered targetwhich is insensitive to the inhibitor. A few positive selection methodsare also known in the art.

One commonly used selectable marker gene for plant transformation is theneomycin phosphotransferase II (nptII) gene which, when under thecontrol of plant regulatory signals, confers resistance to kanamycin.Fraley et al., Proc. Natl. Acad. Sci. USA, 80:4803 (1983). Anothercommonly used selectable marker gene is the hygromycinphosphotransferase gene which confers resistance to the antibiotichygromycin. Vanden Elzen et al., Plant Mol. Biol., 5:299 (1985).

Additional selectable marker genes of bacterial origin that conferresistance to antibiotics include gentamycin acetyl transferase,streptomycin phosphotransferase and aminoglycoside-3′-adenyltransferase, the bleomycin resistance determinant (Hayford et al., PlantPhysiol. 86:1216 (1988); Jones et al., Mol. Gen. Genet., 210:86 (1987);Svab et al., Plant Mol. Biol. 14:197 (1990); Hille et al., Plant Mol.Biol. 7:171 (1986)). Other selectable marker genes confer resistance toherbicides such as glyphosate, glufosinate or bromoxynil (Comai et al.,Nature 317:741-744 (1985); Gordon-Kamm et al., Plant Cell 2:603-618(1990); and Stalker et al., Science 242:419-423 (1988)).

Selectable marker genes for plant transformation not of bacterial origininclude, for example, mouse dihydrofolate reductase, plant5-enolpyruvylshikimate-3-phosphate synthase and plant acetolactatesynthase (Eichholtz et al., Somatic Cell Mol. Genet. 13:67 (1987); Shahet al., Science 233:478 (1986); Charest et al., Plant Cell Rep. 8:643(1990)).

Another class of marker genes for plant transformation requiresscreening of presumptively transformed plant cells rather than directgenetic selection of transformed cells for resistance to a toxicsubstance such as an antibiotic. These genes are particularly useful toquantify or visualize the spatial pattern of expression of a gene inspecific tissues and are frequently referred to as reporter genesbecause they can be fused to a gene or gene regulatory sequence for theinvestigation of gene expression. Commonly used genes for screeningpresumptively transformed cells include β-glucuronidase (GUS),β-galactosidase, luciferase and chloramphenicol acetyltransferase(Jefferson, R. A., Plant Mol. Biol. Rep. 5:387 (1987); Teeri et al.,EMBO J. 8:343 (1989); Koncz et al., Proc. Natl. Acad. Sci. USA 84:131(1987); DeBlock et al., EMBO J. 3:1681 (1984)).

In vivo methods for visualizing GUS activity that do not requiredestruction of plant tissue are available (Molecular Probes publication2908, IMAGENE GREEN, pp. 1-4 (1993) and Naleway et al., J. Cell Biol.115:151a (1991)). However, these in vivo methods for visualizing GUSactivity have not proven useful for recovery of transformed cellsbecause of low sensitivity, high fluorescent backgrounds and limitationsassociated with the use of luciferase genes as selectable markers.

More recently, a gene encoding Green Fluorescent Protein (GFP) has beenutilized as a marker for gene expression in prokaryotic and eukaryoticcells (Chalfie et al., Science 263:802 (1994)). GFP and mutants of GFPmay be used as screenable markers.

Expression Vectors for Tomato Transformation: Promoters

Genes included in expression vectors must be driven by a nucleotidesequence comprising a regulatory element, for example, a promoter.Several types of promoters are well known in the transformation arts asare other regulatory elements that can be used alone or in combinationwith promoters.

As used herein, “promoter” includes reference to a region of DNAupstream from the start of transcription and involved in recognition andbinding of RNA polymerase and other proteins to initiate transcription.A “plant promoter” is a promoter capable of initiating transcription inplant cells. Examples of promoters under developmental control includepromoters that preferentially initiate transcription in certain tissues,such as leaves, roots, seeds, fibers, xylem vessels, tracheids, orsclerenchyma. Such promoters are referred to as “tissue-preferred.”Promoters that initiate transcription only in a certain tissue arereferred to as “tissue-specific.” A “cell-type” specific promoterprimarily drives expression in certain cell types in one or more organs,for example, vascular cells in roots or leaves. An “inducible” promoteris a promoter which is under environmental control. Examples ofenvironmental conditions that may effect transcription by induciblepromoters include anaerobic conditions or the presence of light.Tissue-specific, tissue-preferred, cell type specific, and induciblepromoters constitute the class of “non-constitutive” promoters. A“constitutive” promoter is a promoter that is active under mostenvironmental conditions.

A. Inducible Promoters—An inducible promoter is operably linked to agene for expression in tomato. Optionally, the inducible promoter isoperably linked to a nucleotide sequence encoding a signal sequencewhich is operably linked to a gene for expression in tomato. With aninducible promoter the rate of transcription increases in response to aninducing agent.

Any inducible promoter can be used in the instant invention. See, Wardet al., Plant Mol. Biol. 22:361-366 (1993). Exemplary induciblepromoters include, but are not limited to, that from the ACEI systemwhich responds to copper (Mett et al., Proc. Natl. Acad. Sci. USA90:4567-4571 (1993)); In2 gene from maize which responds tobenzenesulfonamide herbicide safeners (Hershey et al., Mol. Gen Genetics227:229-237 (1991) and Gatz et al., Mol. Gen. Genetics 243:32-38 (1994))or Tet repressor from Tn10 (Gatz et al., Mol. Gen. Genetics 227:229-237(1991)). A particularly preferred inducible promoter is a promoter thatresponds to an inducing agent to which plants do not normally respond.An exemplary inducible promoter is the inducible promoter from a steroidhormone gene, the transcriptional activity of which is induced by aglucocorticosteroid hormone (Schena et al., Proc. Natl. Acad. Sci. USA88:0421 (1991)).

B. Constitutive Promoters—A constitutive promoter is operably linked toa gene for expression in tomato or the constitutive promoter is operablylinked to a nucleotide sequence encoding a signal sequence which isoperably linked to a gene for expression in tomato.

Many different constitutive promoters can be utilized in the instantinvention. Exemplary constitutive promoters include, but are not limitedto, the promoters from plant viruses such as the 35S promoter from CaMV(Odell et al., Nature 313:810-812 (1985)) and the promoters from suchgenes as rice actin (McElroy et al., Plant Cell 2: 163-171 (1990));ubiquitin (Christensen et al., Plant Mol. Biol. 12:619-632 (1989) andChristensen et al., Plant Mol. Biol. 18:675-689 (1992)); pEMU (Last etal., Theor. Appl. Genet. 81:581-588 (1991)); MAS (Velten et al., EMBO J.3:2723-2730 (1984)) and maize H3 histone (Lepetit et al., Mol. Gen.Genetics 231:276-285 (1992) and Atanassova et al., Plant Journal 2 (3):291-300 (1992)). The ALS promoter, Xba1/Ncol fragment 5′ to the Brassicanapus ALS3 structural gene (or a nucleotide sequence similarity to saidXba1/Ncol fragment), represents a particularly useful constitutivepromoter. See PCT Application WO 96/30530.

C. Tissue-specific or Tissue-preferred Promoters—A tissue-specificpromoter is operably linked to a gene for expression in tomato.Optionally, the tissue-specific promoter is operably linked to anucleotide sequence encoding a signal sequence which is operably linkedto a gene for expression in tomato. Plants transformed with a gene ofinterest operably linked to a tissue-specific promoter produce theprotein product of the transgene exclusively, or preferentially, in aspecific tissue.

Any tissue-specific or tissue-preferred promoter can be utilized in theinstant invention. Exemplary tissue-specific or tissue-preferredpromoters include, but are not limited to, a root-preferred promotersuch as that from the phaseolin gene (Murai et al., Science 23:476-482(1983) and Sengupta-Gopalan et al., Proc. Natl. Acad. Sci. USA82:3320-3324 (1985)); a leaf-specific and light-induced promoter such asthat from cab or rubisco (Simpson et al., EMBO J. 4(11):2723-2729 (1985)and Timko et al., Nature 318:579-582 (1985)); an anther-specificpromoter such as that from LAT52 (Twell et al., Mol. Gen. Genetics217:240-245 (1989)); a pollen-specific promoter such as that from Zm13(Guerrero et al., Mol. Gen. Genetics 244:161-168 (1993)) or amicrospore-preferred promoter such as that from apg (Twell et al., Sex.Plant Reprod. 6:217-224 (1993)).

Signal Sequences for Targeting Proteins to Subcellular Compartments

Transport of a protein produced by transgenes to a subcellularcompartment such as the chloroplast, vacuole, peroxisome, glyoxysome,cell wall or mitochondrion or for secretion into the apoplast, isaccomplished by means of operably linking the nucleotide sequenceencoding a signal sequence to the 5′ and/or 3′ region of a gene encodingthe protein of interest. Targeting sequences at the 5′ and/or 3′ end ofthe structural gene may determine during protein synthesis andprocessing where the encoded protein is ultimately compartmentalized.

The presence of a signal sequence directs a polypeptide to either anintracellular organelle or subcellular compartment or for secretion tothe apoplast. Many signal sequences are known in the art. See, forexample, Becker et al., Plant Mol. Biol. 20:49 (1992); Knox, C., et al.,Plant Mol. Biol. 9:3-17 (1987); Lerner et al., Plant Physiol. 91:124-129(1989); Frontes et al., Plant Cell 3:483-496 (1991); Matsuoka et al.,Proc. Natl. Acad. Sci. 88:834 (1991); Gould et al., J. Cell. Biol.108:1657 (1989); Creissen et al., Plant J. 2:129 (1991); Kalderon, etal., Cell 39:499-509 (1984); Steifel, et al., Plant Cell 2:785-793(1990).

Foreign Protein Genes and Agronomic Genes

With transgenic plants according to the present invention, a foreignprotein can be produced in commercial quantities. Thus, techniques forthe selection and propagation of transformed plants, which are wellunderstood in the art, yield a plurality of transgenic plants which areharvested in a conventional manner, and a foreign protein then can beextracted from a tissue of interest or from total biomass. Proteinextraction from plant biomass can be accomplished by known methods whichare discussed, for example, by Heney and Orr, Anal. Biochem. 114:92-6(1981).

According to a preferred embodiment, the transgenic plant provided forcommercial production of foreign protein is a tomato plant. For therelatively small number of transgenic plants that show higher levels ofexpression, a genetic map can be generated, primarily via conventionalRFLP, PCR, and SSR analysis, which identifies the approximatechromosomal location of the integrated DNA molecule. For exemplarymethodologies in this regard, see Glick and Thompson, Methods in PlantMolecular Biology and Biotechnology, CRC Press, Boca Raton 269:284(1993). Map information concerning chromosomal location is useful forproprietary protection of a subject transgenic plant.

Wang et al. discuss “Large Scale Identification, Mapping and Genotypingof Single-Nucleotide Polymorphisms in the Human Genome” Science,280:1077-1082 (1998), and similar capabilities are becoming increasinglyavailable for the tomato genome. Map information concerning chromosomallocation is useful for proprietary protection of a subject transgenicplant. If unauthorized propagation is undertaken and crosses made withother germplasm, the map of the integration region can be compared tosimilar maps for suspect plants to determine if the latter have a commonparentage with the subject plant. Map comparisons would involvehybridizations, RFLP, PCR, SSR, and sequencing, all of which areconventional techniques. SNPs may also be used alone or in combinationwith other techniques.

Likewise, by means of the present invention, plants can be geneticallyengineered to express various phenotypes of horticultural interest.Through the transformation of tomato the expression of genes can bealtered to enhance disease resistance, insect resistance, herbicideresistance, horticultural quality, and other traits. Transformation canalso be used to insert DNA sequences which control or help control malesterility. DNA sequences native to tomato as well as non-native DNAsequences can be transformed into tomato and used to alter levels ofnative or non-native proteins. Various promoters, targeting sequences,enhancing sequences, and other DNA sequences can be inserted into thegenome for the purpose of altering the expression of proteins. Reductionof the activity of specific genes (also known as gene silencing, or genesuppression) is desirable for several aspects of genetic engineering inplants.

Many techniques for gene silencing are well known to one of skill in theart, including, but not limited to, knock-outs (such as by insertion ofa transposable element such as mu (Chandler, V., The Maize Handbook, ch.118, Springer-Verlag (1994)) or other genetic elements such as a FRT,Lox or other site specific integration site, antisense technology (see,e.g., Sheehy et al., PNAS USA 85:8805-8809 (1988); and U.S. Pat. Nos.5,107,065; 5,453,566; and 5,759,829); co-suppression (e.g., Taylor,Plant Cell 9:1245 (1997); Jorgensen, Trends Biotech. 8(12):340-344(1990); Flavell, PNAS USA 91:3490-3496 (1994); Finnegan et al.,Bio/Technology 12: 883-888 (1994); and Neuhuber et al., Mol. Gen. Genet.244:230-241 (1994)); RNA interference (Napoli et al., Plant Cell2:279-289 (1990); U.S. Pat. No. 5,034,323; Sharp, Genes Dev. 13:139-141(1999); Zamore et al., Cell 101:25-33 (2000); and Montgomery et al.,PNAS USA 95:15502-15507 (1998)); virus-induced gene silencing (Burton,et al., Plant Cell 12:691-705 (2000); and Baulcombe, Curr. Op. PlantBio. 2:109-113 (1999)); target-RNA-specific ribozymes (Haseloff et al.,Nature 334: 585-591 (1988)); hairpin structures (Smith et al., Nature407:319-320 (2000); WO 99/53050; and WO 98/53083); MicroRNA (Aukerman &Sakai, Plant Cell 15:2730-2741 (2003)); ribozymes (Steinecke et al.,EMBO J. 11:1525 (1992); and Perriman et al., Antisense Res. Dev. 3:253(1993)); oligonucleotide mediated targeted modification (e.g., WO03/076574 and WO 99/25853); Zn-finger targeted molecules (e.g., WO01/52620; WO 03/048345; and WO 00/42219); and other methods orcombinations of the above methods known to those of skill in the art.

Likewise, by means of the present invention, other genes can beexpressed in transformed plants. More particularly, plants can begenetically engineered to express various phenotypes of interest.Exemplary genes implicated in this regard include, but are not limitedto, those categorized below:

1. Genes that Confer Resistance to Pests or Disease and that Encode:

A. Plant disease resistance genes. Plant defenses are often activated byspecific interaction between the product of a disease resistance gene(R) in the plant and the product of a corresponding avirulence (Avr)gene in the pathogen. A plant variety can be transformed with one ormore cloned resistance genes to engineer plants that are resistant tospecific pathogen strains. See, for example, Jones et al., Science266:789 (1994) (cloning of the tomato Cf-9 gene for resistance toCladosporium fulvum); Martin et al., Science 262:1432 (1993) (tomato Ptogene for resistance to Pseudomonas syringae pv. tomato encodes a proteinkinase); Mindrinos et al. Cell 78:1089 (1994) (Arabidopsis RSP2 gene forresistance to Pseudomonas syringae); McDowell & Woffenden, TrendsBiotechnol. 21(4): 178-83 (2003); and Toyoda et al., Transgenic Res. 11(6):567-82 (2002).

B. A gene conferring resistance to a pest, such as a nematode. See,e.g., PCT Application WO 96/30517; PCT Application WO 93/19181.

C. A Bacillus thuringiensis protein, a derivative thereof or a syntheticpolypeptide modeled thereon. See, for example, Geiser et al., Gene48:109 (1986), who disclose the cloning and nucleotide sequence of a Btδ-endotoxin gene. Moreover, DNA molecules encoding δ-endotoxin genes canbe purchased from American Type Culture Collection, Manassas, Va., forexample, under ATCC Accession Nos. 40098, 67136, 31995, and 31998.

D. A lectin. See, for example, Van Damme et al., Plant Molec. Biol.24:25 (1994), who disclose the nucleotide sequences of several Cliviaminiata mannose-binding lectin genes.

E. A vitamin-binding protein such as avidin. See PCT Application US93/06487 which teaches the use of avidin and avidin homologues aslarvicides against insect pests.

F. An enzyme inhibitor, for example, a protease or proteinase inhibitoror an amylase inhibitor. See, for example, Abe et al., J. Biol. Chem.262:16793 (1987) (nucleotide sequence of rice cysteine proteinaseinhibitor); Huub et al., Plant Molec. Biol. 21:985 (1993) (nucleotidesequence of cDNA encoding tobacco proteinase inhibitor I); Sumitani etal., Biosci. Biotech. Biochem. 57:1243 (1993) (nucleotide sequence ofStreptomyces nitrosporeus α-amylase inhibitor); and U.S. Pat. No.5,494,813 (Hepher and Atkinson, issued Feb. 27, 1996).

G. An insect-specific hormone or pheromone such as an ecdysteroid orjuvenile hormone, a variant thereof, a mimetic based thereon, or anantagonist or agonist thereof. See, for example, the disclosure byHammock et al., Nature 344:458 (1990), of baculovirus expression ofcloned juvenile hormone esterase, an inactivator of juvenile hormone.

H. An insect-specific peptide or neuropeptide which, upon expression,disrupts the physiology of the affected pest. For example, see thedisclosures of Regan, J. Biol. Chem. 269:9 (1994) (expression cloningyields DNA coding for insect diuretic hormone receptor) and Pratt etal., Biochem. Biophys. Res. Comm. 163:1243 (1989) (an allostatin isidentified in Diploptera puntata); Chattopadhyay et al., CriticalReviews in Microbiology 30 (1): 33-54 2004 (2004); Zjawiony, J Nat Prod67 (2): 300-310 (2004) Carlini & Grossi-de-Sa, Toxicon, 40 (11):1515-1539 (2002); Ussuf et al., Curr Sci. 80 (7): 847-853 (2001); andVasconcelos & Oliveira, Toxicon 44 (4): 385-403 (2004). See also, U.S.Pat. No. 5,266,317 to Tomalski et al., which discloses genes encodinginsect-specific, paralytic neurotoxins.

I. An insect-specific venom produced in nature by a snake, a wasp, etc.For example, see, Pang et al., Gene 116:165 (1992), for disclosure ofheterologous expression in plants of a gene coding for a scorpioninsectotoxic peptide.

J. An enzyme responsible for a hyperaccumulation of a monoterpene, asesquiterpene, a steroid, hydroxamic acid, a phenylpropanoid derivativeor another non-protein molecule with insecticidal activity.

K. An enzyme involved in the modification, including thepost-translational modification, of a biologically active molecule; forexample, a glycolytic enzyme, a proteolytic enzyme, a lipolytic enzyme,a nuclease, a cyclase, a transaminase, an esterase, a hydrolase, aphosphatase, a kinase, a phosphorylase, a polymerase, an elastase, achitinase, and a glucanase, whether natural or synthetic. See, PCTApplication WO 93/02197 (Scott et al.), which discloses the nucleotidesequence of a callase gene. DNA molecules which containchitinase-encoding sequences can be obtained, for example, from the ATCCunder Accession Nos. 39637 and 67152. See also, Kramer et al., InsectBiochem. Molec. Biol. 23:691 (1993), who teach the nucleotide sequenceof a cDNA encoding tobacco hornworm chitinase; and Kawalleck et al.,Plant Molec. Biol. 21:673 (1993), who provide the nucleotide sequence ofthe parsley ubi4-2 polyubiquitin gene; U.S. Pat. Nos. 7,145,060;7,087,810; and 6,563,020.

L. A molecule that stimulates signal transduction. For example, see thedisclosure by Botella et al., Plant Molec. Biol. 24:757 (1994), ofnucleotide sequences for mung bean calmodulin cDNA clones, and Griess etal., Plant Physiol. 104:1467 (1994), who provide the nucleotide sequenceof a maize calmodulin cDNA clone.

M. A hydrophobic moment peptide. See PCT Application WO 95/16776 andU.S. Pat. No. 5,580,852, which disclose peptide derivatives oftachyplesin which inhibit fungal plant pathogens, and PCT Application WO95/18855 and U.S. Pat. No. 5,607,914 which teaches syntheticantimicrobial peptides that confer disease resistance.

N. A membrane permease, a channel former, or a channel blocker. Forexample, see the disclosure of Jaynes et al., Plant Sci 89:43 (1993), ofheterologous expression of a cecropin-β lytic peptide analog to rendertransgenic tobacco plants resistant to Pseudomonas solanacearum.

O. A viral-invasive protein or a complex toxin derived therefrom. Forexample, the accumulation of viral coat proteins in transformed plantcells imparts resistance to viral infection and/or disease developmenteffected by the virus from which the coat protein gene is derived, aswell as by related viruses. See, Beachy et al., Ann. Rev. Phytopathol.28:451 (1990). Coat protein-mediated resistance has been conferred upontransformed plants against alfalfa mosaic virus, cucumber mosaic virus,and tobacco mosaic virus.

P. An insect-specific antibody or an immunotoxin derived therefrom.Thus, an antibody targeted to a critical metabolic function in theinsect gut would inactivate an affected enzyme, killing the insect. See,Taylor et al., Abstract #497, Seventh Int'l Symposium on MolecularPlant-Microbe Interactions (Edinburgh, Scotland) (1994) (enzymaticinactivation in transgenic tobacco via production of single-chainantibody fragments).

Q. A virus-specific antibody. See, for example, Tavladoraki et al.,Nature 366:469 (1993), who show that transgenic plants expressingrecombinant antibody genes are protected from virus attack.

R. A developmental-arrestive protein produced in nature by a pathogen ora parasite. Thus, fungal endo-α-1,4-D-polygalacturonases facilitatefungal colonization and plant nutrient release by solubilizing plantcell wall homo-α-1,4-D-galacturonase. See, Lamb et al., Bio/Technology10:1436 (1992). The cloning and characterization of a gene which encodesa bean endopolygalacturonase-inhibiting protein is described by Toubartet al., Plant J. 2:367 (1992).

S. A developmental-arrestive protein produced in nature by a plant. Forexample, Logemann et al., Bio/Technology 10:305 (1992), have shown thattransgenic plants expressing the barley ribosome-inactivating gene havean increased resistance to fungal disease.

T. Genes involved in the Systemic Acquired Resistance (SAR) Responseand/or the pathogenesis-related genes. Briggs, S., Current Biology, 5(2)(1995); Pieterse & Van Loon, Curr. Opin. Plant Bio. 7(4):456-64 (2004);and Somssich, Cell 113(7):815-6 (2003).

U. Antifungal genes. See, Cornelissen and Melchers, Plant Physiol.,101:709-712 (1993); Parijs et al., Planta 183:258-264 (1991); andBushnell et al., Can. J. of Plant Path. 20(2):137-149 (1998). Also see,U.S. Pat. No. 6,875,907.

V. Detoxification genes, such as for fumonisin, beauvericin,moniliformin, and zearalenone, and their structurally relatedderivatives. For example, see, U.S. Pat. No. 5,792,931.

W. Cystatin and cysteine proteinase inhibitors. See, U.S. Pat. No.7,205,453.

X. Defensin genes. See, WO 03/000863 and U.S. Pat. No. 6,911,577.

Y. Genes conferring resistance to nematodes. See, e.g., PCT ApplicationWO 96/30517; PCT Application WO 93/19181; WO 03/033651; Urwin et al.,Planta 204:472-479 (1998); Williamson, Curr Opin Plant Bio. 2(4):327-31(1999).

Z. Genes that confer resistance to Phytophthora root rot, such as theRps 1, Rps 1-a, Rps 1-b, Rps 1-c, Rps 1-d, Rps 1-e, Rps 1-k, Rps 2, Rps3-a, Rps 3-b, Rps 3-c, Rps 4, Rps 5, Rps 6, Rps 7, and other Rps genes.See, for example, Shoemaker et al., “Phytophthora Root Rot ResistanceGene Mapping in Soybean” Plant Genome IV Conference, San Diego, Calif.(1995).

AA. Genes that confer resistance to brown stem rot, such as described inU.S. Pat. No. 5,689,035 and incorporated by reference for this purpose.

2. Genes that Confer Resistance to an Herbicide, for Example:

A. An herbicide that inhibits the growing point or meristem, such as animidazolinone or a sulfonylurea. Exemplary genes in this category codefor mutant ALS and AHAS enzyme as described, for example, by Lee et al.,EMBO J. 7:1241 (1988) and Miki et al., Theor. Appl. Genet. 80:449(1990), respectively.

B. Glyphosate (resistance conferred by mutant5-enolpyruvlshikimate-3-phosphate synthase (EPSPS) and aroA genes,respectively) and other phosphono compounds such as glufosinate(phosphinothricin acetyl transferase (PAT) and Streptomyceshygroscopicus PAT bar genes), and pyridinoxy or phenoxy proprionic acidsand cyclohexanediones (ACCase inhibitor-encoding genes). See, forexample, U.S. Pat. No. 4,940,835 to Shah, et al., which discloses thenucleotide sequence of a form of EPSPS which can confer glyphosateresistance. U.S. Pat. No. 5,627,061 to Barry et al., also describesgenes encoding EPSPS enzymes. See also, U.S. Pat. Nos. 6,566,587;6,338,961; 6,248,876; 6,040,497; 5,804,425; 5,633,435; 5,145,783;4,971,908; 5,312,910; 5,188,642; 4,940,835; 5,866,775; 6,225,114;6,130,366; 5,310,667; 4,535,060; 4,769,061; 5,633,448; 5,510,471; RE36,449; RE 37,287; and 5,491,288; and International PublicationsEP1173580; WO 01/66704; EP1173581; and EP1173582, which are incorporatedherein by reference for this purpose. Glyphosate resistance is alsoimparted to plants that express a gene that encodes a glyphosateoxido-reductase enzyme as described more fully in U.S. Pat. Nos.5,776,760 and 5,463,175, which are incorporated herein by reference forthis purpose. In addition glyphosate resistance can be imparted toplants by the over expression of genes encoding glyphosateN-acetyltransferase. See, for example, U.S. application Ser. No.10/427,692. A DNA molecule encoding a mutant aroA gene can be obtainedunder ATCC Accession No. 39256, and the nucleotide sequence of themutant gene is disclosed in U.S. Pat. No. 4,769,061 to Comai. EuropeanPatent Application No. 0 333 033 to Kumada et al., and U.S. Pat. No.4,975,374 to Goodman et al., disclose nucleotide sequences of glutaminesynthetase genes which confer resistance to herbicides such asL-phosphinothricin. The nucleotide sequence of a PAT gene is provided inEuropean Application No. 0 242 246 to Leemans et al. DeGreef et al.,Bio/Technology 7:61 (1989) describe the production of transgenic plantsthat express chimeric bar genes coding for phosphinothricin acetyltransferase activity. Exemplary of genes conferring resistance tophenoxy proprionic acids and cyclohexones, such as sethoxydim andhaloxyfop are the Acc1-S1, Acc1-S2, and Acc2-S3 genes described byMarshall et al., Theor. Appl. Genet. 83:435 (1992).

C. An herbicide that inhibits photosynthesis, such as a triazine (psbAand gs+ genes) and a benzonitrile (nitrilase gene). Przibila et al.,Plant Cell 3:169 (1991), describe the transformation of Chlamydomonaswith plasmids encoding mutant psbA genes. Nucleotide sequences fornitrilase genes are disclosed in U.S. Pat. No. 4,810,648 to Stalker andDNA molecules containing these genes are available under ATCC AccessionNos. 53435, 67441, and 67442. Cloning and expression of DNA coding for aglutathione S-transferase is described by Hayes et al., Biochem. J.285:173 (1992).

D. Acetohydroxy acid synthase, which has been found to make plants thatexpress this enzyme resistant to multiple types of herbicides, has beenintroduced into a variety of plants. See, Hattori et al., Mol. Gen.Genet. 246:419 (1995). Other genes that confer tolerance to herbicidesinclude a gene encoding a chimeric protein of rat cytochrome P4507A1 andyeast NADPH-cytochrome P450 oxidoreductase (Shiota et al., PlantPhysiol., 106:17 (1994); genes for glutathione reductase and superoxidedismutase (Aono et al., Plant Cell Physiol. 36:1687 (1995); and genesfor various phosphotransferases (Datta et al., Plant Mol. Biol. 20:619(1992).

E. Protoporphyrinogen oxidase (protox) is necessary for the productionof chlorophyll, which is necessary for all plant survival. The protoxenzyme serves as the target for a variety of herbicidal compounds. Theseherbicides also inhibit growth of all the different species of plantspresent, causing their total destruction. The development of plantscontaining altered protox activity which are resistant to theseherbicides are described in U.S. Pat. Nos. 6,288,306; 6,282,837;5,767,373; and International Publication WO 01/12825.

3. Genes that Confer or Contribute to a Value-Added Trait, Such as:

A. Modified fatty acid metabolism, for example, by transforming a plantwith an antisense gene of stearyl-ACP desaturase to increase stearicacid content of the plant. See, Knultzon et al., Proc. Natl. Acad. Sci.USA 89:2625 (1992).

B. Decreased phytate content—1) Introduction of a phytase-encoding geneenhances breakdown of phytate, adding more free phosphate to thetransformed plant. For example, see, Van Hartingsveldt et al., Gene127:87 (1993), for a disclosure of the nucleotide sequence of anAspergillus niger phytase gene. 2) Up-regulation of a gene that reducesphytate content. This, for example, could be accomplished, by cloningand then re-introducing DNA associated with one or more of the alleles,such as the LPA alleles, identified in maize mutants characterized bylow levels of phytic acid, such as in Raboy et al., Maydica 35: 383(1990) and/or by altering inositol kinase activity as in WO 02/059324;U.S. Publ. No. 2003/000901; WO 03/027243; U.S. Publ No. 2003/0079247; WO99/05298; U.S. Pat. Nos. 6,197,561; 6,291,224; 6,391,348; WO2002/059324; U.S. Publ. No. 2003/0079247; WO 98/45448; WO 99/55882; WO01/04147.

C. Modified carbohydrate composition effected, for example, bytransforming plants with a gene coding for an enzyme that alters thebranching pattern of starch, or a gene altering thioredoxin such as NTRand/or TRX (see, U.S. Pat. No. 6,531,648, which is incorporated byreference for this purpose) and/or a gamma zein knock out or mutant suchas cs27, or TUSC27, or en27 (see, U.S. Pat. No. 6,858,778 and U.S. Publ.Nos. 2005/0160488; 2005/0204418, which are incorporated by reference forthis purpose). See, Shiroza et al., J. Bacteriol. 170: 810 (1988)(nucleotide sequence of Streptococcus mutans fructosyltransferase gene);Steinmetz et al., Mol. Gen. Genet. 200: 220 (1985) (nucleotide sequenceof Bacillus subtilis levansucrase gene); Pen et al., Bio/Technology 10:292 (1992) (production of transgenic plants that express Bacilluslicheniformis alpha-amylase); Elliot et al., Plant Molec. Biol. 21: 515(1993) (nucleotide sequences of tomato invertase genes); Sogaard et al.,J. Biol. Chem. 268: 22480 (1993) (site-directed mutagenesis of barleyalpha-amylase gene); and Fisher et al., Plant Physiol. 102: 1045 (1993)(maize endosperm starch branching enzyme II); WO 99/10498 (improveddigestibility and/or starch extraction through modification ofUDP-D-xylose 4-epimerase, Fragile 1 and 2, Ref 1, HCHL, C4H); U.S. Pat.No. 6,232,529 (method of producing high oil seed by modification ofstarch levels (AGP)). The fatty acid modification genes mentioned abovemay also be used to affect starch content and/or composition through theinterrelationship of the starch and oil pathways.

D. Altered antioxidant content or composition, such as alteration oftocopherol or tocotrienols. For example, see, U.S. Pat. Nos. 6,787,683and 7,154,029 and WO 00/68393 involving the manipulation of antioxidantlevels through alteration of a phytl prenyl transferase (ppt); WO03/082899 through alteration of a homogentisate geranyl transferase(hggt).

4. Genes that Control Male Sterility.

There are several methods of conferring genetic male sterilityavailable, such as multiple mutant genes at separate locations withinthe genome that confer male sterility, as disclosed in U.S. Pat. Nos.4,654,465 and 4,727,219 to Brar et al. and chromosomal translocations asdescribed by Patterson in U.S. Pat. Nos. 3,861,709 and 3,710,511. Inaddition to these methods, Albertsen et al., U.S. Pat. No. 5,432,068,describe a system of nuclear male sterility which includes: identifyinga gene which is critical to male fertility; silencing this native genewhich is critical to male fertility; removing the native promoter fromthe essential male fertility gene and replacing it with an induciblepromoter; inserting this genetically engineered gene back into theplant; and thus creating a plant that is male sterile because theinducible promoter is not “on” resulting in the male fertility gene notbeing transcribed. Fertility is restored by inducing, or turning “on,”the promoter, which in turn allows the gene that confers male fertilityto be transcribed.

A. Introduction of a deacetylase gene under the control of atapetum-specific promoter and with the application of the chemicalN-Ac-PPT. See International Publication WO 01/29237.

B. Introduction of various stamen-specific promoters. See InternationalPublications WO 92/13956 and WO 92/13957.

C. Introduction of the barnase and the barstar genes. See Paul et al.,Plant Mol. Biol. 19:611-622 (1992).

For additional examples of nuclear male and female sterility systems andgenes, see also, U.S. Pat. Nos. 5,859,341; 6,297,426; 5,478,369;5,824,524; 5,850,014; and 6,265,640, all of which are herebyincorporated by reference.

5. Genes that Create a Site for Site Specific DNA Integration.

This includes the introduction of FRT sites that may be used in theFLP/FRT system and/or Lox sites that may be used in the Cre/Loxp system.For example, see Lyznik, et al., Site-Specific Recombination for GeneticEngineering in Plants, Plant Cell Rep 21:925-932 (2003) and WO 99/25821,which are hereby incorporated by reference. Other systems that may beused include the Gin recombinase of phage Mu (Maeser et al. (1991);Chandler, V., The Maize Handbook, ch. 118, Springer-Verlag (1994); thePin recombinase of E. coli (Enomoto et al. (1983)), and the R/RS systemof the pSR1 plasmid (Araki et al. (1992).

6. Genes that Affect Abiotic Stress Resistance.

Genes that affect abiotic stress resistance (including, but not limitedto, flowering and fruit development, drought resistance or tolerance,cold resistance or tolerance, and salt resistance or tolerance) andincreased yield under stress. For example, see, WO 00/73475 where wateruse efficiency is altered through alteration of malate; U.S. Pat. Nos.5,892,009; 5,965,705; 5,929,305; 5,891,859; 6,417,428; 6,664,446;6,706,866; 6,717,034; 6,801,104; WO 2000/060089; WO 2001/026459; WO2001/035725; WO 2001/034726; WO 2001/035727; WO 2001/036444; WO2001/036597; WO 2001/036598; WO 2002/015675; WO 2002/017430; WO2002/077185; WO 2002/079403; WO 2003/013227; WO 2003/013228; WO2003/014327; WO 2004/031349; WO 2004/076638; WO 98/09521; and WO99/38977 describing genes, including CBF genes and transcription factorseffective in mitigating the negative effects of freezing, high salinity,and drought on plants, as well as conferring other positive effects onplant phenotype; U.S. Publ. No. 2004/0148654 and WO 01/36596 whereabscisic acid is altered in plants resulting in improved plant phenotypesuch as increased yield and/or increased tolerance to abiotic stress; WO2000/006341, WO 04/090143, U.S. application Ser. No. 10/817,483 and U.S.Pat. No. 6,992,237 where cytokinin expression is modified resulting inplants with increased stress tolerance, such as drought tolerance,and/or increased yield. Also see, WO 02/02776; WO 2003/052063;JP2002281975; U.S. Pat. No. 6,084,153; WO 01/64898; U.S. Pat. Nos.6,177,275 and 6,107,547 (enhancement of nitrogen utilization and alterednitrogen responsiveness). For ethylene alteration, see, U.S. Publ. Nos.2004/0128719; 2003/0166197 and WO 2000/32761. For plant transcriptionfactors or transcriptional regulators of abiotic stress, see, e.g., U.S.Publ. Nos. 2004/0098764 or 2004/0078852.

Other genes and transcription factors that affect plant growth and othertraits such as yield, flowering, plant growth, and/or plant structure,can be introduced or introgressed into plants, see, e.g., WO 97/49811;WO 98/56918; WO 97/10339; and U.S. Pat. Nos. 6,573,430; 6,713,663; WO96/14414; WO 96/38560; WO 01/21822; WO 00/44918; WO 99/49064; WO00/46358; WO 97/29123; U.S. Pat. Nos. 6,794,560; 6,307,126, WO 99/09174(D8 and Rht); WO 2004/076638 and WO 2004/031349 (transcription factors).

Methods for Tomato Transformation

Numerous methods for plant transformation have been developed includingbiological and physical plant transformation protocols. See, forexample, Miki et al., “Procedures for Introducing Foreign DNA intoPlants” in Methods in Plant Molecular Biology and Biotechnology, Glick,B. R. and Thompson, J. E. Eds. (CRC Press, Inc. Boca Raton (1993)) pp.67-88. In addition, expression vectors and in-vitro culture methods forplant cell or tissue transformation and regeneration of plants areavailable. See, for example, Gruber et al., “Vectors for PlantTransformation” in Methods in Plant Molecular Biology and Biotechnology,Glick, B. R. and Thompson, J. E. Eds. (CRC Press, Inc., Boca Raton(1993)) pp. 89-119.

A. Agrobacterium-mediated Transformation—One method for introducing anexpression vector into plants is based on the natural transformationsystem of Agrobacterium. See, for example, Horsch et al., Science227:1229 (1985). A. tumefaciens and A. rhizogenes are plant pathogenicsoil bacteria which genetically transform plant cells. The Ti and Riplasmids of A. tumefaciens and A. rhizogenes, respectively, carry genesresponsible for genetic transformation of the plant. See, for example,Kado, C. I., Crit. Rev. Plant Sci. 10:1 (1991). Descriptions ofAgrobacterium vector systems and methods for Agrobacterium-mediated genetransfer are provided by Gruber et al., supra, Miki et al., supra andMoloney et al., Plant Cell Reports 8:238 (1989). See also, U.S. Pat. No.5,563,055 (Townsend and Thomas, issued Oct. 8, 1996).

B. Direct Gene Transfer—Several methods of plant transformation,collectively referred to as direct gene transfer, have been developed asan alternative to Agrobacterium-mediated transformation. A generallyapplicable method of plant transformation is microprojectile-mediatedtransformation where DNA is carried on the surface of microprojectilesmeasuring 1 to 4 μm. The expression vector is introduced into planttissues with a biolistic device that accelerates the microprojectiles tospeeds of 300 to 600 m/s which is sufficient to penetrate plant cellwalls and membranes. Sanford et al., Part. Sci. Technol. 5:27 (1987);Sanford, J. C., Trends Biotech. 6:299 (1988); Klein et al., Bio/Tech.6:559-563 (1988); Sanford, J. C. Physiol Plant 7:206 (1990); Klein etal., Biotechnology 10:268 (1992). See also, U.S. Pat. No. 5,015,580(Christou, et al., issued May 14, 1991) and U.S. Pat. No. 5,322,783(Tomes, et al., issued Jun. 21, 1994).

Another method for physical delivery of DNA to plants is sonication oftarget cells. Zhang et al., Bio/Technology 9:996 (1991). Alternatively,liposome and spheroplast fusion have been used to introduce expressionvectors into plants. Deshayes et al., EMBO J., 4:2731 (1985); Christouet al., Proc Natl. Acad. Sci. USA 84:3962 (1987). Direct uptake of DNAinto protoplasts using CaCl₂ precipitation, polyvinyl alcohol orpoly-L-ornithine has also been reported. Hain et al., Mol. Gen. Genet.199:161 (1985) and Draper et al., Plant Cell Physiol. 23:451 (1982).Electroporation of protoplasts and whole cells and tissues have alsobeen described (Donn et al., In Abstracts of VIIth InternationalCongress on Plant Cell and Tissue Culture IAPTC, A2-38, p. 53 (1990);D'Halluin et al., Plant Cell 4:1495-1505 (1992) and Spencer et al.,Plant Mol. Biol. 24:51-61 (1994)).

Following transformation of tomato target tissues, expression of theabove-described selectable marker genes allows for preferentialselection of transformed cells, tissues and/or plants, usingregeneration and selection methods well known in the art.

The foregoing methods for transformation would typically be used forproducing a transgenic variety. The transgenic variety could then becrossed with another (non-transformed or transformed) variety in orderto produce a new transgenic variety. Alternatively, a genetic trait thathas been engineered into a particular tomato line using the foregoingtransformation techniques could be moved into another line usingtraditional backcrossing techniques that are well known in the plantbreeding arts. For example, a backcrossing approach could be used tomove an engineered trait from a public, variety into a desirable hybrid,or from a variety containing a foreign gene in its genome into a varietyor varieties that do not contain that gene. As used herein, “crossing”can refer to a simple X by Y cross or the process of backcrossingdepending on the context.

Genetic Marker Profile Through SSR and First Generation Progeny

In addition to phenotypic observations, a plant can also be identifiedby its genotype. The genotype of a plant can be characterized through agenetic marker profile which can identify plants of the same variety ora related variety or be used to determine or validate a pedigree.Genetic marker profiles can be obtained by techniques such asRestriction Fragment Length Polymorphisms (RFLPs), Randomly AmplifiedPolymorphic DNAs (RAPDs), Arbitrarily Primed Polymerase Chain Reaction(AP-PCR), DNA Amplification Fingerprinting (DAF), Sequence CharacterizedAmplified Regions (SCARs), Amplified Fragment Length Polymorphisms(AFLPs), Simple Sequence Repeats (SSRs) which are also referred to asMicrosatellites, and Single Nucleotide Polymorphisms (SNPs). Forexample, see, Cregan et. al, “An Integrated Genetic Linkage Map of theSoybean Genome” Crop Science 39:1464-1490 (1999) and Berry et al.,Assessing Probability of Ancestry Using Simple Sequence Repeat Profiles:Applications to Maize Inbred Lines and Soybean Varieties” Genetics165:331-342 (2003), each of which are incorporated by reference hereinin their entirety.

Particular markers used for these purposes are not limited to anyparticular set of markers, but are envisioned to include any type ofmarker and marker profile which provides a means of distinguishingvarieties. One method of comparison is to use only homozygous loci fortomato hybrid ‘Vespolino.’

The present invention comprises a tomato plant characterized bymolecular and physiological data obtained from the representative sampleof said variety deposited with the American Type Culture Collection(ATCC). Further provided by the invention is a tomato plant formed bythe combination of the disclosed tomato plant or plant cell with anothertomato plant or cell and comprising the homozygous alleles of thevariety.

Means of performing genetic marker profiles using SSR polymorphisms arewell known in the art. SSRs are genetic markers based on polymorphismsin repeated nucleotide sequences, such as microsatellites. A markersystem based on SSRs can be highly informative in linkage analysisrelative to other marker systems in that multiple alleles may bepresent. Another advantage of this type of marker is that, through useof flanking primers, detection of SSRs can be achieved, for example, bythe polymerase chain reaction (PCR), thereby eliminating the need forlabor-intensive Southern hybridization. The PCR detection is done by useof two oligonucleotide primers flanking the polymorphic segment ofrepetitive DNA. Repeated cycles of heat denaturation of the DNA followedby annealing of the primers to their complementary sequences at lowtemperatures, and extension of the annealed primers with DNA polymerase,comprise the major part of the methodology.

Following amplification, markers can be scored by electrophoresis of theamplification products. Scoring of marker genotype is based on the sizeof the amplified fragment, which may be measured by the number of basepairs of the fragment. While variation in the primer used or inlaboratory procedures can affect the reported fragment size, relativevalues should remain constant regardless of the specific primer orlaboratory used. When comparing varieties it is preferable if all SSRprofiles are performed in the same lab.

The SSR profile of tomato hybrid ‘Vespolino’ can be used to identifyplants comprising ‘Vespolino’ as a parent, since such plants willcomprise the same homozygous alleles as ‘Vespolino.’ Because the tomatovariety is essentially homozygous at all relevant loci, most loci shouldhave only one type of allele present. In contrast, a genetic markerprofile of an F₁ progeny should be the sum of those parents, e.g., ifone parent was homozygous for allele x at a particular locus, and theother parent homozygous for allele y at that locus, then the F₁ progenywill be xy (heterozygous) at that locus. Subsequent generations ofprogeny produced by selection and breeding are expected to be ofgenotype x (homozygous), y (homozygous), or xy (heterozygous) for thatlocus position. When the F₁ plant is selfed or sibbed for successivefilial generations, the locus should be either x or y for that position.

In addition, plants and plant parts substantially benefiting from theuse of ‘Vespolino’ in their development, such as ‘Vespolino’ comprisinga backcross conversion, transgene, or genetic sterility factor, may beidentified by having a molecular marker profile with a high percentidentity to ‘Vespolino.’ Such a percent identity might be 95%, 96%, 97%,98%, 99%, 99.5%, or 99.9% identical to ‘Vespolino.’

The SSR profile of ‘Vespolino’ also can be used to identify essentiallyderived varieties and other progeny varieties developed from the use of‘Vespolino,’ as well as cells and other plant parts thereof. Such plantsmay be developed using the markers identified in WO 00/31964; U.S. Pat.No. 6,162,967; and U.S. application Ser. No. 09/954,773. Progeny plantsand plant parts produced using ‘Vespolino’ may be identified by having amolecular marker profile of at least 25%, 30%, 35%, 40%, 45%, 50%, 55%,60%, 65%, 70%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%,86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or99.5% genetic contribution from tomato variety, as measured by eitherpercent identity or percent similarity. Such progeny may be furthercharacterized as being within a pedigree distance of ‘Vespolino,’ suchas within 1, 2, 3, 4, or 5 or less cross-pollinations to a tomato plantother than ‘Vespolino’ or a plant that has ‘Vespolino’ as a progenitor.Unique molecular profiles may be identified with other molecular toolssuch as SNPs and RFLPs.

While determining the SSR genetic marker profile of the plants describedsupra, several unique SSR profiles may also be identified which did notappear in either parent of such plant. Such unique SSR profiles mayarise during the breeding process from recombination or mutation. Acombination of several unique alleles provides a means of identifying aplant variety, an F₁ progeny produced from such variety, and progenyproduced from such variety.

Gene Conversions

When the term “tomato plant” is used in the context of the presentinvention, this also includes any single gene conversions of thatvariety. The term gene converted plant as used herein refers to thosetomato plants which are developed by a plant breeding technique calledbackcrossing wherein essentially all of the desired morphological andphysiological characteristics of a variety are recovered in addition tothe one or more genes transferred into the variety via the backcrossingtechnique. Backcrossing methods can be used with the present inventionto improve or introduce a characteristic into the variety. The term“backcrossing” as used herein refers to the repeated crossing of ahybrid progeny back to the recurrent parent, i.e., backcrossing 1, 2, 3,4, 5, 6, 7, 8, or more times to the recurrent parent. The parentaltomato plant that contributes the gene for the desired characteristic istermed the nonrecurrent or donor parent. This terminology refers to thefact that the nonrecurrent parent is used one time in the backcrossprotocol and therefore does not recur. The parental tomato plant towhich the gene or genes from the nonrecurrent parent are transferred isknown as the recurrent parent as it is used for several rounds in thebackcrossing protocol (Poehlman & Sleper (1994); Fehr, Principles ofCultivar Development, pp. 261-286 (1987)). In a typical backcrossprotocol, the original variety of interest (recurrent parent) is crossedto a second variety (nonrecurrent parent) that carries the single geneof interest to be transferred. The resulting progeny from this cross arethen crossed again to the recurrent parent and the process is repeateduntil a tomato plant is obtained wherein essentially all of the desiredmorphological and physiological characteristics of the recurrent parentare recovered in the converted plant, in addition to the singletransferred gene from the nonrecurrent parent.

The selection of a suitable recurrent parent is an important step for asuccessful backcrossing procedure. The goal of a backcross protocol isto alter or substitute a single trait or characteristic in the originalvariety. To accomplish this, a single gene of the recurrent variety ismodified or substituted with the desired gene from the nonrecurrentparent, while retaining essentially all of the rest of the desiredgenetic, and therefore the desired physiological and morphological,constitution of the original variety. The choice of the particularnonrecurrent parent will depend on the purpose of the backcross; one ofthe major purposes is to add some agronomically important trait to theplant. The exact backcrossing protocol will depend on the characteristicor trait being altered to determine an appropriate testing protocol.Although backcrossing methods are simplified when the characteristicbeing transferred is a dominant allele, a recessive allele may also betransferred. In this instance it may be necessary to introduce a test ofthe progeny to determine if the desired characteristic has beensuccessfully transferred.

Many single gene traits have been identified that are not regularlyselected for in the development of a new variety but that can beimproved by backcrossing techniques. Single gene traits may or may notbe transgenic; examples of these traits include but are not limited to,male sterility, herbicide resistance, resistance for bacterial, fungal,or viral disease, insect resistance, male fertility, enhancednutritional quality, industrial usage, yield stability, and yieldenhancement. These genes are generally inherited through the nucleus.Several of these single gene traits are described in U.S. Pat. Nos.5,959,185; 5,973,234; and 5,977,445, the disclosures of which arespecifically hereby incorporated by reference.

Introduction of a New Trait or Locus into ‘Vespolino’

‘Vespolino’ represents a new base genetic variety into which a new locusor trait may be introgressed. Direct transformation and backcrossingrepresent two important methods that can be used to accomplish such anintrogression. The term backcross conversion and single locus conversionare used interchangeably to designate the product of a backcrossingprogram.

Backcross Conversions of ‘Vespolino’

A backcross conversion of ‘Vespolino’ occurs when DNA sequences areintroduced through backcrossing (Hallauer et al., “Corn Breeding” Cornand Corn Improvements, No. 18, pp. 463-481 (1988), with ‘Vespolino’utilized as the recurrent parent. Both naturally occurring andtransgenic DNA sequences may be introduced through backcrossingtechniques. A backcross conversion may produce a plant with a trait orlocus conversion in at least two or more backcrosses, including at leasttwo crosses, at least three crosses, at least four crosses, at leastfive crosses and the like. Molecular marker assisted breeding orselection may be utilized to reduce the number of backcrosses necessaryto achieve the backcross conversion. For example, see, Openshaw, S. J.et al., Marker-assisted Selection in Backcross Breeding. In: ProceedingsSymposium of the Analysis of Molecular Data, Crop Science Society ofAmerica, Corvallis, Oreg. (August 1994), where it is demonstrated that abackcross conversion can be made in as few as two backcrosses.

The complexity of the backcross conversion method depends on the type oftrait being transferred (single genes or closely linked genes as vs.unlinked genes), the level of expression of the trait, the type ofinheritance (cytoplasmic or nuclear), and the types of parents includedin the cross. It is understood by those of ordinary skill in the artthat for single gene traits that are relatively easy to classify, thebackcross method is effective and relatively easy to manage. (See,Hallauer et al. in Corn and Corn Improvement, Sprague and Dudley, ThirdEd. (1998)). Desired traits that may be transferred through backcrossconversion include, but are not limited to, sterility (nuclear andcytoplasmic), fertility restoration, nutritional enhancements, droughttolerance, nitrogen utilization, industrial enhancements, diseaseresistance (bacterial, fungal or viral), insect resistance, andherbicide resistance. In addition, an introgression site itself, such asan FRT site, Lox site or other site specific integration site, may beinserted by backcrossing and utilized for direct insertion of one ormore genes of interest into a specific plant variety. In someembodiments of the invention, the number of loci that may be backcrossedinto ‘Vespolino’ is at least 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10. A singlelocus may contain several transgenes, such as a transgene for diseaseresistance that, in the same expression vector, also contains atransgene for herbicide resistance. The gene for herbicide resistancemay be used as a selectable marker and/or as a phenotypic trait. Asingle locus conversion of site specific integration system allows forthe integration of multiple genes at the converted loci.

The backcross conversion may result from either the transfer of adominant allele or a recessive allele. Selection of progeny containingthe trait of interest is accomplished by direct selection for a traitassociated with a dominant allele. Transgenes transferred viabackcrossing typically function as a dominant single gene trait and arerelatively easy to classify. Selection of progeny for a trait that istransferred via a recessive allele requires growing and selfing thefirst backcross generation to determine which plants carry the recessivealleles. Recessive traits may require additional progeny testing insuccessive backcross generations to determine the presence of the locusof interest. The last backcross generation is usually selfed to givepure breeding progeny for the gene(s) being transferred, although abackcross conversion with a stably introgressed trait may also bemaintained by further backcrossing to the recurrent parent withselection for the converted trait.

Along with selection for the trait of interest, progeny are selected forthe phenotype of the recurrent parent. The backcross is a form ofinbreeding, and the features of the recurrent parent are automaticallyrecovered after successive backcrosses. Poehlman, Breeding Field Crops,p. 204 (1987). Poehlman suggests from one to four or more backcrosses,but as noted above, the number of backcrosses necessary can be reducedwith the use of molecular markers. Other factors, such as a geneticallysimilar donor parent, may also reduce the number of backcrossesnecessary. As noted by Poehlman, backcrossing is easiest for simplyinherited, dominant and easily recognized traits.

One process for adding or modifying a trait or locus in ‘Vespolino’comprises crossing ‘Vespolino’ plants grown from ‘Vespolino’ seed withplants of another tomato variety that comprise the desired trait orlocus, selecting F₁ progeny plants that comprise the desired trait orlocus to produce selected F₁ progeny plants, crossing the selectedprogeny plants with the ‘Vespolino’ plants to produce backcross progenyplants, selecting for backcross progeny plants that have the desiredtrait or locus and the morphological characteristics of ‘Vespolino’ toproduce selected backcross progeny plants, and backcrossing to‘Vespolino’ two, three or more times in succession to produce selectedthird, fourth or higher backcross progeny plants that comprise saidtrait or locus. The modified ‘Vespolino’ may be further characterized ashaving the physiological and morphological characteristics of‘Vespolino’ listed in Table 1 as determined at the 5% significance levelwhen grown in the same environmental conditions and/or may becharacterized by percent similarity or identity to ‘Vespolino’ asdetermined by SSR markers. The above method may be utilized with fewerbackcrosses in appropriate situations, such as when the donor parent ishighly related or markers are used in the selection step. Desired traitsthat may be used include those nucleic acids known in the art, some ofwhich are listed herein, that will affect traits through nucleic acidexpression or inhibition. Desired loci include the introgression of FRT,Lox and other sites for site specific integration, which may also affecta desired trait if a functional nucleic acid is inserted at theintegration site.

In addition, the above process and other similar processes describedherein may be used to produce first generation progeny tomato seed byadding a step at the end of the process that comprises crossing‘Vespolino’ with the introgressed trait or locus with a different tomatoplant and harvesting the resultant first generation progeny tomato seed.

Tissue Culture

Further reproduction of the variety can occur by tissue culture andregeneration. Tissue culture of various tissues of tomatoes andregeneration of plants therefrom is well known and widely published. Forexample, reference may be had to Girish-Chandel et al., Advances inPlant Sciences 13: 1, 11-17 (2000); Costa et al., Plant Cell Report 19:3 327-332 (2000); Plastira et al., Acta Horticulturae 447, 231-234(1997); Zagorska et al., Plant Cell Report 17: 12 968-973 (1998);Asahura et al., Breeding Science 45: 455-459 (1995); Chen et al.,Breeding Science 44: 3, 257-262 (1994): Patil et al., Plant and Tissueand Organ Culture 36: 2, 255-258 (1994); Gill, R., et al., SomaticEmbryogenesis and Plant Regeneration from Seedling Cultures of Tomato(Lycopersicon esculentum Mill.), J. Plant Physiol. 147:273-276 (1995);José M. Seguí-Simarro and Fernando Nuez, Embryogenesis induction,callogenesis, and plant regeneration by in vitro culture of tomatoisolated microspores and whole anthers J. Exp. Bot. 58: 1119-1132 (March2007); Hamza et al., Re-evaluation of Conditions for Plant Regenerationand Agrobacterium-Mediated Transformation from Tomato (Lycopersiconesculentum), J. Exp. Bot. 44: 1837-1845 (December 1993). Thus, anotheraspect of this invention is to provide cells which upon growth anddifferentiation produce tomato plants having the physiological andmorphological characteristics of ‘Vespolino.’

As used herein, the term “tissue culture” indicates a compositioncomprising isolated cells of the same or a different type or acollection of such cells organized into parts of a plant. Exemplarytypes of tissue cultures are protoplasts, calli, plant clumps, and plantcells that can generate tissue culture that are intact in plants orparts of plants, such as embryos, pollen, flowers, seeds, fruit,petioles, leaves, stems, roots, root tips, anthers, pistils, and thelike. Means for preparing and maintaining plant tissue culture are wellknown in the art. By way of example, a tissue culture comprising organshas been used to produce regenerated plants. U.S. Pat. Nos. 5,959,185;5,973,234; and 5,977,445 describe certain techniques, the disclosures ofwhich are incorporated herein by reference.

Using ‘Vespolino’ to Develop Other Tomato Varieties

Tomato varieties such as ‘Vespolino’ are typically developed for use asfresh produce or for processing. However, tomato varieties such as‘Vespolino’ also provide a source of breeding material that may be usedto develop new tomato varieties. Plant breeding techniques known in theart and used in a tomato plant breeding program include, but are notlimited to, recurrent selection, mass selection, bulk selection, massselection, backcrossing, pedigree breeding, open pollination breeding,restriction fragment length polymorphism enhanced selection, geneticmarker enhanced selection, making double haploids, and transformation.Often combinations of these techniques are used. The development oftomato varieties in a plant breeding program requires, in general, thedevelopment and evaluation of homozygous varieties. There are manyanalytical methods available to evaluate a new variety. The oldest andmost traditional method of analysis is the observation of phenotypictraits but genotypic analysis may also be used.

Additional Breeding Methods

This invention is directed to methods for producing a tomato plant bycrossing a first parent tomato plant with a second parent tomato plantwherein either the first or second parent tomato plant is ‘Vespolino.’The other parent may be any other tomato plant, such as a tomato plantthat is part of a synthetic or natural population. Any such methodsusing ‘Vespolino’ are part of this invention: selfing, sibbing,backcrosses, mass selection, pedigree breeding, bulk selection, hybridproduction, crosses to populations, and the like. These methods are wellknown in the art and some of the more commonly used breeding methods aredescribed below. Descriptions of breeding methods can be found in one ofseveral reference books (e.g., Allard, Principles of Plant Breeding(1960); Simmonds, Principles of Crop Improvement (1979); Sneep et al.(1979); Fehr, “Breeding Methods for Cultivar Development,” 2.sup.nd ed.,Wilcox editor (1987).

The following describes breeding methods that may be used with‘Vespolino’ in the development of further tomato plants. One suchembodiment is a method for developing a ‘Vespolino’ progeny tomato plantin a tomato plant breeding program comprising: obtaining the tomatoplant, or a part thereof, of ‘Vespolino’ utilizing said plant or plantpart as a source of breeding material and selecting a ‘Vespolino’progeny plant with molecular markers in common with ‘Vespolino’ and/orwith morphological and/or physiological characteristics selected fromthe characteristics listed in Table 1. Breeding steps that may be usedin the tomato plant breeding program include pedigree breeding,backcrossing, mutation breeding, and recurrent selection. In conjunctionwith these steps, techniques such as RFLP-enhanced selection, geneticmarker enhanced selection (for example, SSR markers), and the making ofdouble haploids may be utilized.

Another method involves producing a population of ‘Vespolino’ progenytomato plants, comprising crossing ‘Vespolino’ with another tomatoplant, thereby producing a population of tomato plants, which, onaverage, derive 50% of their alleles from ‘Vespolino.’ A plant of thispopulation may be selected and repeatedly selfed or sibbed with a tomatocultivar resulting from these successive filial generations. Oneembodiment of this invention is the tomato cultivar produced by thismethod and that has obtained at least 50% of its alleles from‘Vespolino.’

One of ordinary skill in the art of plant breeding would know how toevaluate the traits of two plant varieties to determine if there is nosignificant difference between the two traits expressed by thosevarieties. For example, see, Fehr and Walt, Principles of CultivarDevelopment, pp. 261-286 (1987). Thus the invention includes‘Vespolino’progeny tomato plants comprising a combination of at leasttwo hybrid ‘Vespolino’ traits selected from the group consisting ofthose listed in Tables 1 and 2 or the ‘Vespolino’ combination of traitslisted in the Summary of the Invention, so that said progeny tomatoplant is not significantly different for said traits than ‘Vespolino’.Using techniques described herein, molecular markers may be used toidentify said progeny plant as a ‘Vespolino’ progeny plant. Mean traitvalues may be used to determine whether trait differences aresignificant, and preferably the traits are measured on plants grownunder the same environmental conditions. Once such a variety isdeveloped its value is substantial since it is important to advance thegermplasm base as a whole in order to maintain or improve traits such asyield, disease resistance, pest resistance, and plant performance inextreme environmental conditions.

Progeny of ‘Vespolino’ may also be characterized through their filialrelationship with ‘Vespolino,’ as for example, being within a certainnumber of breeding crosses of ‘Vespolino.’ A breeding cross is a crossmade to introduce new genetics into the progeny, and is distinguishedfrom a cross, such as a self or a sib cross, made to select amongexisting genetic alleles. The lower the number of breeding crosses inthe pedigree, the closer the relationship between ‘Vespolino’ and itsprogeny. For example, progeny produced by the methods described hereinmay be within 1, 2, 3, 4, or 5 breeding crosses of ‘Vespolino.’

As used herein, the term “plant” includes plant cells, plantprotoplasts, plant cell tissue cultures from which tomato plants can beregenerated, plant calli, plant clumps, and plant cells that are intactin plants or parts of plants, such as embryos, pollen, ovules, flowers,fruit, leaves, roots, root tips, anthers, cotyledons, hypocotyls,meristematic cells, stems, pistils, petiole, and the like.

Pedigree Breeding

Pedigree breeding starts with the crossing of two genotypes, such as‘Vespolino’ and another tomato variety having one or more desirablecharacteristics that is lacking or which complements ‘Vespolino.’ If thetwo original parents do not provide all the desired characteristics,other sources can be included in the breeding population. In thepedigree method, superior plants are selfed and selected in successivefilial generations. In the succeeding filial generations theheterozygous condition gives way to homogeneous varieties as a result ofself-pollination and selection. Typically in the pedigree method ofbreeding, five or more successive filial generations of selfing andselection is practiced: F₁ to F₂; F₂ to F₃; F₃ to F₄; F₄ to F₅; etc.After a sufficient amount of inbreeding, successive filial generationswill serve to increase seed of the developed variety. Preferably, thedeveloped variety comprises homozygous alleles at about 95% or more ofits loci.

In addition to being used to create a backcross conversion, backcrossingcan also be used in combination with pedigree breeding. As discussedpreviously, backcrossing can be used to transfer one or morespecifically desirable traits from one variety, the donor parent, to adeveloped variety called the recurrent parent, which has overall goodcharacteristics yet lacks that desirable trait or traits. However, thesame procedure can be used to move the progeny toward the genotype ofthe recurrent parent but at the same time retain many components of thenonrecurrent parent by stopping the backcrossing at an early stage andproceeding with selfing and selection. For example, a tomato variety maybe crossed with another variety to produce a first generation progenyplant. The first generation progeny plant may then be backcrossed to oneof its parent varieties to create a BC₁ or BC₂. Progeny are selfed andselected so that the newly developed variety has many of the attributesof the recurrent parent and yet several of the desired attributes of thenon-recurrent parent. This approach leverages the value and strengths ofthe recurrent parent for use in new tomato varieties.

Therefore, an embodiment of this invention is a method of making abackcross conversion of ‘Vespolino,’ comprising the steps of crossing aplant of ‘Vespolino’ with a donor plant comprising a desired trait,selecting an F₁ progeny plant comprising the desired trait, andbackcrossing the selected F₁ progeny plant to a plant of ‘Vespolino.’This method may further comprise the step of obtaining a molecularmarker profile of ‘Vespolino’ and using the molecular marker profile toselect for a progeny plant with the desired trait and the molecularmarker profile of ‘Vespolino.’ In one embodiment the desired trait is amutant gene or transgene present in the donor parent.

Recurrent Selection and Mass Selection

Recurrent selection is a method used in a plant breeding program toimprove a population of plants. ‘Vespolino’ is suitable for use in arecurrent selection program. The method entails individual plants crosspollinating with each other to form progeny. The progeny are grown andthe superior progeny selected by any number of selection methods, whichinclude individual plant, half-sib progeny, full-sib progeny and selfedprogeny. The selected progeny are cross pollinated with each other toform progeny for another population. This population is planted andagain superior plants are selected to cross pollinate with each other.Recurrent selection is a cyclical process and therefore can be repeatedas many times as desired. The objective of recurrent selection is toimprove the traits of a population. The improved population can then beused as a source of breeding material to obtain new varieties forcommercial or breeding use, including the production of a syntheticcultivar. A synthetic cultivar is the resultant progeny formed by theintercrossing of several selected varieties.

Mass selection is a useful technique when used in conjunction withmolecular marker enhanced selection. In mass selection seeds fromindividuals are selected based on phenotype or genotype. These selectedseeds are then bulked and used to grow the next generation. Bulkselection requires growing a population of plants in a bulk plot,allowing the plants to self-pollinate, harvesting the seed in bulk, andthen using a sample of the seed harvested in bulk to plant the nextgeneration. Also, instead of self pollination, directed pollinationcould be used as part of the breeding program.

Mutation Breeding

Mutation breeding is another method of introducing new traits into‘Vespolino.’ Mutations that occur spontaneously or are artificiallyinduced can be useful sources of variability for a plant breeder. Thegoal of artificial mutagenesis is to increase the rate of mutation for adesired characteristic. Mutation rates can be increased by manydifferent means including temperature, long-term seed storage, tissueculture conditions, radiation; such as X-rays, Gamma rays (e.g., cobalt60 or cesium 137), neutrons (product of nuclear fission by uranium 235in an atomic reactor), Beta radiation (emitted from radioisotopes suchas phosphorus 32 or carbon 14), or ultraviolet radiation (preferablyfrom 2500 to 2900 nm), or chemical mutagens (such as base analogues(5-bromo-uracil)), related compounds (8-ethoxy caffeine), antibiotics(streptonigrin), alkylating agents (sulfur mustards, nitrogen mustards,epoxides, ethylenamines, sulfates, sulfonates, sulfones, lactones),azide, hydroxylamine, nitrous acid, or acridines. Once a desired traitis observed through mutagenesis the trait may then be incorporated intoexisting germplasm by traditional breeding techniques. Details ofmutation breeding can be found in Fehr, “Principles of CultivarDevelopment” Macmillan Publishing Company (1993). In addition, mutationscreated in other tomato plants may be used to produce a backcrossconversion of ‘Vespolino’ that comprises such mutation.

Breeding with Molecular Markers

Molecular markers, which includes markers identified through the use oftechniques such as Isozyme Electrophoresis, Restriction Fragment LengthPolymorphisms (RFLPs), Randomly Amplified Polymorphic DNAs (RAPDs),Arbitrarily Primed Polymerase Chain Reaction (AP-PCR), DNA AmplificationFingerprinting (DAF), Sequence Characterized Amplified Regions (SCARs),Amplified Fragment Length Polymorphisms (AFLPs), Simple Sequence Repeats(SSRs), and Single Nucleotide Polymorphisms (SNPs), may be used in plantbreeding methods utilizing ‘Vespolino.’

Isozyme Electrophoresis and RFLPs have been widely used to determinegenetic composition. Shoemaker and Olsen, “Molecular Linkage Map ofSoybean” (Glycine max L. Merr.), pp. 6.131-6.138 (1993). In S. J.O'Brien (ed.) Genetic Maps: Locus Maps of Complex Genomes, Cold SpringHarbor Laboratory Press, Cold Spring Harbor, N.Y., developed a moleculargenetic linkage map that consisted of 25 linkage groups with about 365RFLP, 11 RAPD (random amplified polymorphic DNA), three classicalmarkers, and four isozyme loci. See also, Shoemaker, R. C., “RFLP Map ofSoybean” pp. 299-309 (1994). In R. L. Phillips and I. K. Vasil (ed.)DNA-based markers in plants, Kluwer Academic Press Dordrecht, theNetherlands.

SSR technology is currently the most efficient and practical markertechnology; more marker loci can be routinely used and more alleles permarker locus can be found using SSRs in comparison to RFLPs. Forexample, Diwan and Cregan described a highly polymorphic microsatelliteloci in tomato with as many as 26 alleles. (Diwan, N., and P. B. Cregan,“Automated sizing of fluorescent-labeled simple sequence repeat (SSR)markers to assay genetic variation in Soybean” Theor. Appl. Genet.95:220-225 (1997)). Single Nucleotide Polymorphisms may also be used toidentify the unique genetic composition of the invention and progenyvarieties retaining that unique genetic composition. Various molecularmarker techniques may be used in combination to enhance overallresolution.

One use of molecular markers is Quantitative Trait Loci (QTL) mapping.QTL mapping is the use of markers, which are known to be closely linkedto alleles that have measurable effects on a quantitative trait.Selection in the breeding process is based upon the accumulation ofmarkers linked to the positive effecting alleles and/or the eliminationof the markers linked to the negative effecting alleles from the plant'sgenome.

Molecular markers can also be used during the breeding process for theselection of qualitative traits. For example, markers closely linked toalleles or markers containing sequences within the actual alleles ofinterest can be used to select plants that contain the alleles ofinterest during a backcrossing breeding program. The markers can also beused to select for the genome of the recurrent parent and against thegenome of the donor parent. Using this procedure can minimize the amountof genome from the donor parent that remains in the selected plants. Itcan also be used to reduce the number of crosses back to the recurrentparent needed in a backcrossing program. The use of molecular markers inthe selection process is often called genetic marker enhanced selection.Molecular markers may also be used to identify and exclude certainsources of germplasm as parental varieties or ancestors of a plant byproviding a means of tracking genetic profiles through crosses.

Production of Double Haploids

The production of double haploids can also be used for the developmentof plants with a homozygous phenotype in the breeding program. Forexample, a tomato plant for which ‘Vespolino’ is a parent can be used toproduce double haploid plants. Double haploids are produced by thedoubling of a set of chromosomes (1N) from a heterozygous plant toproduce a completely homozygous individual. For example, see, Wan etal., “Efficient Production of Doubled Haploid Plants Through ColchicineTreatment of Anther-Derived Maize Callus” Theoretical and AppliedGenetics, 77:889-892 (1989) and U.S. Pat. No. 7,135,615. This can beadvantageous because the process omits the generations of selfing neededto obtain a homozygous plant from a heterozygous source.

Haploid induction systems have been developed for various plants toproduce haploid tissues, plants and seeds. The haploid induction systemcan produce haploid plants from any genotype by crossing a selected line(as female) with an inducer line. Such inducer lines for maize includeStock 6 (Coe, Am. Nat. 93:381-382 (1959); Sharkar and Coe, Genetics54:453-464 (1966); KEMS (Deimling, Roeber, and Geiger, Vortr.Pflanzenzuchtg 38:203-224 (1997); or KMS and ZMS (Chalyk, Bylich &Chebotar, MNL 68:47 (1994); Chalyk & Chebotar, Plant Breeding119:363-364 (2000); and indeterminate gametophyte (ig) mutation(Kermicle, Science 166:1422-1424 (1969), the disclosures of which areincorporated herein by reference.

Methods for obtaining haploid plants are also disclosed in Kobayashi,M., et al., J. Heredity 71(1):9-14 (1980); Pollacsek, M., Agronomie(Paris) 12(3):247-251 (1992); Cho-Un-Haing et al., J. Plant Biol.39(3):185-188 (1996); Chalyk et al., Maize Genet Coop. Newsletter 68:47(1994).

Thus, an embodiment of this invention is a process for making asubstantially homozygous ‘Vespolino’ progeny plant by producing orobtaining a seed from the cross of ‘Vespolino’ and another tomato plantand applying double haploid methods to the F₁ seed or F₁ plant or to anysuccessive filial generation. Based on studies in maize and currentlybeing conducted in tomato, such methods would decrease the number ofgenerations required to produce a variety with similar genetics orcharacteristics to ‘Vespolino.’ See, Bernardo, R. and Kahler, A. L.,Theor. Appl. Genet. 102:986-992 (2001).

In particular, a process of making seed retaining the molecular markerprofile of ‘Vespolino’ is contemplated, such process comprisingobtaining or producing F₁ seed for which ‘Vespolino’ is a parent,inducing doubled haploids to create progeny without the occurrence ofmeiotic segregation, obtaining the molecular marker profile of‘Vespolino,’ and selecting progeny that retain the molecular markerprofile of ‘Vespolino.’

Descriptions of other breeding methods that are commonly used fordifferent traits and crops can be found in one of several referencebooks (e.g., Allard (1960); Simmonds (1979); Sneep et al. (1979); Fehr(1987)).

Deposit Information

A deposit of the ENZA ZADEN BEHEER B.V. proprietary tomato hybrid‘Vespolino’ disclosed above and recited in the appended claims has beenmade with the American Type Culture Collection (ATCC), 10801 UniversityBoulevard, Manassas, Va. 20110. The date of deposit was Apr. 13, 2010.The deposit of 2,500 seeds was taken from the same deposit maintained byENZA ZADEN BEHEER B.V. since prior to the filing date of thisapplication. All restrictions will be removed upon granting of a patent,and the deposit is intended to meet all of the requirements of 37 C.F.R.§§1.801-1.809. The ATCC Accession Number is PTA-10808. The deposit willbe maintained in the depository for a period of thirty years, or fiveyears after the last request, or for the enforceable life of the patent,whichever is longer, and will be replaced as necessary during thatperiod.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity andunderstanding. However, it will be obvious that certain changes andmodifications such as single gene modifications and mutations,somaclonal variants, variant individuals selected from large populationsof the plants of the instant hybrid and the like may be practiced withinthe scope of the invention, as limited only by the scope of the appendedclaims.

1. A seed of tomato hybrid ‘Vespolino,’ representative sample seed ofsaid hybrid was deposited under ATCC Accession No. PTA-10808.
 2. Atomato plant, or a part thereof, produced by growing the seed ofclaim
 1. 3. A tissue culture produced from protoplasts or cells from theplant of claim 2, wherein said cells or protoplasts of the tissueculture are produced from a plant part selected from the groupconsisting of leaf, pollen, embryo, cotyledon, hypocotyl, meristematiccell, root, root tip, pistil, anther, flower, shoot, shoot tip, stem,fruit and petiole.
 4. A tomato plant regenerated from the tissue cultureof claim 3, wherein the plant has all of the morphological andphysiological characteristics of hybrid ‘Vespolino.’
 5. A tomato fruitof the plant of claim
 2. 6. A method for producing a tomato seedcomprising crossing two tomato plants and harvesting the resultanttomato seed, wherein at least one tomato plant is the tomato plant ofclaim
 2. 7. A method of vegetatively propagating a tomato plantcomprising: a) collecting part of the plant of claim 2; b) obtaining aplantlet from said part; and c) growing a plant from said plantlet.
 8. Atomato plant, or a part thereof, produced by the plant of claim
 7. 9. Atomato fruit produced by the plant of claim
 8. 10. A method forproducing a tomato plant that contains in its genetic material one ormore transgenes, wherein the method comprises crossing the tomato plantof claim 2 with a transformed tomato plant of the hybrid tomato‘Vespolino,’ so that the genetic material of the progeny that resultsfrom the cross contains the transgene(s) operably linked to a regulatoryelement and wherein the transgene confers a trait selected from thegroup consisting of male sterility, male fertility, herbicideresistance, insect resistance, and disease resistance.
 11. A method ofproducing an herbicide resistant tomato plant, wherein the methodcomprises introducing a gene conferring herbicide resistance into thetomato plant of claim 2 wherein the gene confers resistance to anherbicide selected from the group consisting of imidazolinone,cyclohexanedione, sulfonylurea, glyphosate, glufosinate, phenoxyproprionic acid, L-phosphinothricin, triazine and benzonitrile.
 12. Anherbicide resistant tomato plant produced by the method of claim
 11. 13.A method of producing a pest or insect resistant tomato plant, whereinthe method comprises introducing a gene conferring pest or insectresistance into the tomato plant of claim
 2. 14. A pest or insectresistant tomato plant produced by the method of claim
 13. 15. Thetomato plant of claim 14, wherein the gene encodes a Bacillusthuringiensis endotoxin.
 16. A method of producing a disease resistanttomato plant, wherein the method comprises introducing a gene conferringdisease resistance into the tomato plant of claim
 2. 17. A diseaseresistant tomato plant produced by the method of claim
 16. 18. A methodof producing a tomato plant derived from the hybrid tomato variety‘Vespolino’, the method comprising the steps of: (a) preparing a progenyplant derived from hybrid tomato variety ‘Vespolino’ by crossing theplant of claim 2 with a second tomato plant; (b) crossing the progenyplant with itself or a second tomato plant to produce a seed of aprogeny plant of a subsequent generation; (c) growing a progeny plant ofa subsequent generation from said seed and crossing the progeny plant ofa subsequent generation with itself or a second tomato plant; and (d)repeating step b) or c) for at least 1 more generation to produce atomato plant derived from the hybrid tomato variety ‘Vespolino’.